U.S. patent number 11,066,666 [Application Number 15/925,172] was granted by the patent office on 2021-07-20 for bioactive renal cells.
This patent grant is currently assigned to inRegen. The grantee listed for this patent is inRegen. Invention is credited to Joydeep Basu, Timothy A. Bertram, Andrew T. Bruce, Sumana Choudhury, Bryan R. Cox, Christopher W. Genheimer, Kelly I. Guthrie, Craig R. Halberstadt, Roger M. Ilagan, Deepak Jain, Manuel J. Jayo, Russell W. Kelley, Oluwatoyin A. Knight, John W. Ludlow, Darell McCoy, Richard Payne, Sharon C. Presnell, Elias A. Rivera, Neil F. Robins, Namrata D. Sangha, Thomas Spencer, Shay M. Wallace, Benjamin Watts, Eric Werdin.
United States Patent |
11,066,666 |
Bertram , et al. |
July 20, 2021 |
Bioactive renal cells
Abstract
The present invention concerns bioactive renal cells
populations, renal cell constructs, and methods of making and using
the same.
Inventors: |
Bertram; Timothy A. (George
Town, KY), Ilagan; Roger M. (Burlington, NC),
Kelley; Russell W. (Winston-Salem, NC), Presnell; Sharon
C. (Lewisville, NC), Choudhury; Sumana (Kernersville,
NC), Bruce; Andrew T. (Lexington, NC), Genheimer;
Christopher W. (Colfax, NC), Cox; Bryan R.
(Winston-Salem, NC), Guthrie; Kelly I. (Winston-Salem,
NC), Basu; Joydeep (Winston-Salem, NC), Wallace; Shay
M. (Winston-Salem, NC), Werdin; Eric (Lewisville,
TX), Knight; Oluwatoyin A. (Winston-Salem, NC), Sangha;
Namrata D. (Winston-Salem, NC), Ludlow; John W.
(Carrboro, NC), Halberstadt; Craig R. (Clemmons, NC),
Payne; Richard (Winston-Salem, NC), Robins; Neil F.
(Winston-Salem, NC), McCoy; Darell (Clemmons, NC), Jain;
Deepak (Winston-Salem, NC), Jayo; Manuel J.
(Winston-Salem, NC), Rivera; Elias A. (Oak Ridge, NC),
Spencer; Thomas (Winston-Salem, NC), Watts; Benjamin
(King, NC) |
Applicant: |
Name |
City |
State |
Country |
Type |
inRegen |
Grand Cayman |
N/A |
KY |
|
|
Assignee: |
inRegen (Grand Cayman,
KY)
|
Family
ID: |
1000005685613 |
Appl.
No.: |
15/925,172 |
Filed: |
March 19, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180282726 A1 |
Oct 4, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13697206 |
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10077442 |
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PCT/US2011/036347 |
May 12, 2011 |
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61473111 |
Apr 7, 2011 |
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61441423 |
Feb 10, 2011 |
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61412933 |
Nov 12, 2010 |
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61413382 |
Nov 12, 2010 |
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61388765 |
Oct 1, 2010 |
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61376586 |
Aug 24, 2010 |
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61372077 |
Aug 9, 2010 |
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61371888 |
Aug 9, 2010 |
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61353895 |
Jun 11, 2010 |
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61334032 |
May 12, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/113 (20130101); C12Q 1/6883 (20130101); C12N
5/0686 (20130101); C12Q 2600/178 (20130101); C12N
2310/141 (20130101); C12N 2320/32 (20130101); C12Q
2600/106 (20130101); C12N 2320/30 (20130101) |
Current International
Class: |
A61K
48/00 (20060101); C12N 5/071 (20100101); C12N
15/113 (20100101); C12Q 1/6883 (20180101); C07H
21/04 (20060101) |
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Jun 2015 |
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WO |
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2017/178472 |
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Oct 2017 |
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WO |
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2017/191234 |
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Nov 2017 |
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WO |
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|
Primary Examiner: Bowman; Amy H
Attorney, Agent or Firm: Mintz Levin Cohn Ferris Glovsky and
Popeo, P.C.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 13/697,206, which is a national stage application, filed under
35 U.S.C. .sctn. 371, of International Application No.
PCT/US11/36347 filed May 12, 2011, which claims the benefit under
35 U.S.C. .sctn. 119 of U.S. Provisional Application No.
61/473,111, filed Apr. 7, 2011; US Provisional Application No.
61/441,423, filed Feb. 10, 2011; U.S. Provisional Application No.
61/413,382, filed Nov. 12, 2010; U.S. Provisional Application No.
61/412,933, filed Nov. 12, 2010; U.S. Provisional Application No.
61/388,765, filed Oct. 1, 2010; U.S. Provisional Application No.
61/376,586, filed Aug. 24, 2010; U.S. Provisional Application No.
61/372,077, filed Aug. 9, 2010; U.S. Provisional Application No.
61/371,888, filed Aug. 9, 2010; U.S. Provisional Application No.
61/353,895, filed Jun. 11, 2010; and U.S. Provisional Application
No. 61/334,032, filed May 12, 2010, the entire contents of each of
which are hereby incorporated by reference herein.
Claims
What is claimed is:
1. A method of providing a regenerative effect to a native kidney
comprising in vivo contacting the native kidney with a composition
comprising isolated human secreted vesicles produced by a renal
cell population enriched for bioactive kidney cells, wherein the
vesicles comprise exosomes or microvesicles comprising a paracrine
factor that attenuates Plasminogen Activation Inhibitor-1 (PAI-1)
and/or Transforming Growth Factor Beta (TGF.beta.) signaling,
wherein the vesicles have been isolated from the renal cell
population, and wherein the regenerative effect comprises a
reduction in renal fibrosis.
2. The method of claim 1, wherein the vesicles comprise
microvesicles.
3. The method of claim 1, wherein the vesicles comprise
exosomes.
4. The method of claim 1, wherein the paracrine factor inhibits
PAI-1 signaling.
5. The method of claim 1, wherein the paracrine factor is an
miRNA.
6. The method of claim 5, wherein the miRNA inhibits PAI-1
signaling.
7. The method of claim 1, wherein the paracrine factor inhibits
TGF.beta..
8. The method of claim 7, wherein the paracrine factor is an miRNA
that inhibits TGF.beta..
9. The method of claim 1, wherein renal fibrosis is reduced by
inhibition of epithelial-to-mesenchymal transition (EMT).
10. The method of claim 1, wherein the bioactive kidney cells
comprise: (a) tubular cells; or (b) tubular cells and one or more
of glomerular cells and vascular cells.
11. The method of claim 10, wherein the bioactive cells comprise
tubular cells and glomerular cells.
12. The method of claim 10, wherein the bioactive cells comprise
tubular cells and vascular cells.
13. The method of claim 1, wherein the renal cell population is
non-autologous to the native kidney.
14. The method of claim 1, wherein the renal cell population is
autologous to the native kidney.
15. The method of claim 6, wherein the miRNA comprises microRNA
30b-5p or microRNA 449a.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted electronically in ASCII text format and is hereby
incorporated by reference in its entirety. Said ASCII text copy,
created on Mar. 19, 2018, is named
"050400_506C01US_Sequence_Listing.txt" and is 1,154 bytes in
size.
FIELD OF THE INVENTION
The present invention relates to bioactive renal cell populations
or fractions that lack cellular components as compared to a healthy
individual yet retain therapeutic properties, and methods of
isolating and culturing the same, as well as methods of treating a
subject in need with the cell populations. In addition, the present
invention relates to methods of providing regenerative effects to a
native kidney using bioactive renal cell populations.
BACKGROUND OF THE INVENTION
Chronic Kidney Disease (CKD) affects over 19 million people in the
United States and is frequently a consequence of metabolic
disorders involving obesity, diabetes, and hypertension.
Examination of the data reveals that the rate of increase is due to
the development of renal failure secondary to hypertension and
non-insulin dependent diabetes mellitus (NIDDM) (United States
Renal Data System: Costs of CKD and ESRD. ed. Bethesda, Md.,
National Institutes of Health, National Institute of Diabetes and
Digestive and Kidney Diseases, 2007 pp 223-238)--two diseases that
are also on the rise worldwide. Obesity, hypertension, and poor
glycemic control have all been shown to be independent risk factors
for kidney damage, causing glomerular and tubular lesions and
leading to proteinuria and other systemically-detectable
alterations in renal filtration function (Aboushwareb, et al.,
World J Urol, 26: 295-300, 2008; Amami, K. et al., Nephrol Dial
Transplant, 13: 1958-66, 1998). CKD patients in stages 1-3 of
progression are managed by lifestyle changes and pharmacological
interventions aimed at controlling the underlying disease state(s),
while patients in stages 4-5 are managed by dialysis and a drug
regimen that typically includes anti-hypertensive agents,
erythropoiesis stimulating agents (ESAs), iron and vitamin D
supplementation. Regenerative medicine technologies may provide
next-generation therapeutic options for CKD. Presnell et al.
WO2010/056328 describe isolated renal cells, including tubular and
erythropoietin (EPO)-producing kidney cell populations, and methods
of isolating and culturing the same, as well as methods of treating
a subject in need with the cell populations. There is a need for
new treatment paradigms that provide substantial and durable
augmentation of kidney functions, to slow progression and improve
quality of life in this patient population.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method for
providing a regenerative effect to a native kidney. In one
embodiment, the method includes the step of in vivo contacting the
native kidney with products secreted by an enriched renal cell
population. In another embodiment, the products are secreted by an
enriched renal cell population that is not part of a construct, as
described herein, e.g., the cell population is not seeded on a
scaffold. In one other embodiment, the products are secreted from a
renal cell construct comprising an enriched renal cell population
directly seeded on or in a scaffold. In another embodiment, the
secretion of the products is bioresponsive to oxygen levels.
Secretion may be induced by a less than atmospheric oxygen level.
In one other embodiment, the lower oxygen level is less than about
5% oxygen.
In one embodiment, the regenerative effect is a reduction in
epithelial-mesenchymal transition (EMT). The reduction in EMT may
be achieved via attenuation of TGF-.beta. signalling and/or
attenuation of Plasminogen Activator Inhibitor-1 (PAI-1)
signalling. In another embodiment, the regenerative effect is a
reduction in renal fibrosis and/or a reduction in renal
inflammation. In some embodiments, the reduction in inflammation
may be mediated by NF.kappa.B. In one other embodiment, the
regenerative effect is characterized by differential expression of
a stem cell marker in the native kidney. The expression may be an
upregulation of marker expression in the in vivo contacted native
kidney relative to expression in a non-contacted native kidney.
In one aspect, the enriched renal cell population includes one or
more cell populations, i.e., an admixture, as described herein. In
one embodiment, the population includes a first cell population,
B2, that contains an enriched population of tubular cells. In
another embodiment, the population includes an admixture of human
renal cells having a first cell population, B2, and a second cell
population, which contains one or more of erythropoietin
(EPO)-producing cells, glomerular cells and vascular cells. In one
other embodiment, the second cell population is a B4 cell
population. In yet another embodiment, the second cell population
is a B3 cell population.
In one embodiment, the admixture further includes a third cell
population having one or more of erythropoietin (EPO)-producing
cells, glomerular cells and vascular cells. In another embodiment,
the third cell population is a B4 cell population. In one other
embodiment, the third cell population is a B3 cell population.
In all embodiments, the B2 cell population has a density between
about 1.045 g/mL and about 1.052 g/mL. In all embodiments, the B4
cell population has a density between about 1.063 g/mL and about
1.091 g/mL. In all embodiments, the B3 cell population has a
density between about 1.052 g/ml and about 1.063 g/ml.
In all embodiments, the enriched renal cell population may be
non-autologous to the native kidney. In all embodiments, the
enriched renal cell population may be autologous to the native
kidney.
In all embodiments, the products include paracrine factors,
endocrine factors, juxtacrine factors, RNA, vesicles,
microvesicles, exosomes and any combination thereof. In one other
embodiment, the vesicles include one or more secreted products
selected from the group consisting of paracrine factors, endocrine
factors, juxtacrine factors, and RNA. In another embodiment, the
products are secreted from a renal cell construct comprising an
enriched renal cell population directly seeded on or in a
scaffold.
In all embodiments, the scaffold may contain a biocompatible
material. In all embodiments, the biocompatible material may be a
hydrogel.
In one embodiment, the present invention provides methods of
assessing whether a kidney disease (KD) patient is responsive to
treatment with a therapeutic. The method may include the step of
determining or detecting the amount of vesicles or their luminal
contents in a test sample obtained from a KD patient treated with
the therapeutic, as compared to or relative to the amount of
vesicles in a control sample, wherein a higher or lower amount of
vesicles or their luminal contents in the test sample as compared
to the amount of vesicles or their luminal contents in the control
sample is indicative of the treated patient's responsiveness to
treatment with the therapeutic. The vesicles may be kidney derived
vesicles. The test sample may contain urine. The vesicles may
contain a biomarker, which may be miRNA. The therapeutic may
contain an enriched population of renal cells.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1B show enrichment of epo-producing cell fraction from
freshly-dissociated kidney tissue using a multi-layered step
gradient technique (FIG. 1A--left panel) or a single-layer mixing
gradient technique (FIG. 1B--right panel). Both methods result in
the partial depletion of non epo-producing cell components
(predominantly tubular cells) from the epo band, which appears
between 1.025 g/mL and 1.035 g/mL.
FIG. 2 shows step gradients of "normoxic" (21% oxygen) and
"hypoxic" (2% oxygen) rodent cultures that were harvested
separately and applied side-by-side to identical step
gradients.
FIG. 3 shows step gradients of "normoxic" (21% oxygen) and
"hypoxic" (2% oxygen) canine cultures that were harvested
separately and applied side-by-side to identical step
gradients.
FIG. 4 shows histopathologic features of the HK17 and HK19
samples.
FIG. 5 shows high content analysis (HCA) of albumin transport in
human NKA cells defining regions of interest (ROI).
FIG. 6 shows quantitative comparison of albumin transport in NKA
cells derived from non-CKD and CKD kidney.
FIG. 7 depicts comparative analysis of marker expression between
tubular-enriched B2 and tubular cell-depleted B4 subtractions.
FIG. 8 depicts comparative functional analysis of albumin transport
between tubular-enriched B2 and tubular cell-depleted B4
subtractions.
FIG. 9 shows expression of SOX2 mRNA in host tissue after treatment
of 5/6 NX rats.
FIG. 10 Western blot showing time course of expression of CD24,
CD133, UTF1, SOX2, NODAL and LEFTY.
FIG. 11 depicts a time course of regenerative response index
(RRI).
FIG. 12 provides a schematic for the preparation and analysis of
UNFX conditioned media.
FIGS. 13A-13D show that conditioned media from UNFX cultures
affects multiple cellular processes in vitro that are potentially
associated with regenerative outcomes. FIG. 13A shows that
UNFX-conditioned media attenuates TNF-a mediated activation of
NF-kB. FIG. 13B shows that UNFX-conditioned media increases
proangiogenic behavior of HUVEC cell cultures. FIG. 13C shows that
UNFX-conditioned media attenuates fibrosis pathways in epithelial
cells. FIG. 13D depicts the positive feedback loop established by
TGF.beta.1 and Plasminogen Activator Inhibitor-1 (PAI-1).
FIGS. 14A-14B show a Western blot analysis demonstrating the
attenuation of fibrosis pathways in mesangial cells.
FIGS. 15A-15C shows that the conditioned media from UNFX contains
secreted vesicles. FIG. 15A depicts secreted vesicles, which are
bilipid structures (red) that encompass cytoplasm-derived internal
components (green). FIGS. 15B-15C show FACS sorting.
FIG. 16A shows a Western blot in which total protein was prepared
and assayed for PAI-1 and bActin. FIG. 16B depicts the microRNA,
miR-30b-5p.
FIGS. 17A-17C show representative immunohistochemistry images of
PAI-1 in Lewis rat kidneys following delivery of bioactive kidney
cells after undergoing a nephrectomy. FIG. 17D shows a comparison
of PAI-1 expression in untreated, nephrectomized rats (red
squares), treated, nephrectomized rats (blue diamonds), and control
animals (green triangles). FIG. 17E shows representative Western
blot analysis on kidney samples taken at 3 and 6 months
post-treatment. FIG. 17F shows a 2-hour exposure to NKA conditioned
media reduces nuclear localization of NF.kappa.B p65. FIG. 17G
depicts the canonical activation of the NFkB pathway by
TNF.alpha..
FIGS. 18A-18B show the nuclear localization of NFkB p65 subunit in
animals with (A) progressive CKD initiated by 5/6 nephrectomy and
(B) non-progressive renal insufficiency initiated by unilateral
nephrectomy. FIGS. 18C-18D show (C) a Western blot analysis for
NFkB p65 in extracts of Lewis rat kidney tissue that have undergone
the 5/6 nephrectomy and (D) electrophoretic mobility shift assay
(EMSA) on extracts. FIG. 18E shows immunohistochemical detection of
the NF.kappa.B p65 subunit in tissue obtained from Lewis rats with
established CKD that received intra-renal injection of NKA (panel
A) or non-bioactive renal cells (panel B).
FIGS. 19A-19C show in vivo evaluation of biomaterials at 1 week and
4 weeks post-implantation.
FIGS. 20A-20D show live/dead staining of NKA constructs. FIGS.
20E-20G show transcriptomic profiling of NKA constructs.
FIGS. 21A-21B show the secretomic profiling of NKA Constructs.
FIGS. 22A-22B show proteomic profiling of NKA Constructs.
FIGS. 23A-23C show confocal microscopy of NKA Constructs.
FIGS. 24A-24B show in vivo evaluation of NKA Constructs at 1 week
and 4 weeks post-implantation.
FIGS. 25A-25D show in vivo evaluation of NKA Construct at 8 weeks
post-implantation.
FIG. 26 shows conditioned medium from NKA Constructs attenuates
TGF-.beta. induced EMT in HK2 cells in vitro.
FIG. 27 depicts the procedure for exposing cells to low oxygen
during processing,
FIG. 28 shows that upon exposure to 2% Oxygen, the following was
observed: alters distribution of cells across a density gradient,
improves overall post-gradient yield
FIG. 29A depicts an assay developed to observe repair of tubular
monolayers in vitro. FIG. 29B shows results of a Quantitative Image
Analysis (BD Pathway 855 BioImager). FIG. 29C shows cells induced
with 2% oxygen to be more proficient at repair of tubular
epithelial monolayers.
FIG. 30A depicts an assay developed to observe repair of tubular
monolayers in vitro. FIG. 30B shows that the induction of cells
with 2% Oxygen enhanced the migration and wound repair compared to
un-induced (21% oxygen). FIG. 30C plots the % of migrated cells
against migration time.
FIGS. 31A-31B show that osteopontin is secreted by tubular cells
and is upregulated in response to injury (Osteopontin
Immunocytochemistry: Hoechst nuclear stain (blue). Osteopontin
(Red), 10.times.). Osteopontin is upregulated by injury in
established tubular cell monolayers as shown by immunofluorescence
(FIG. 31A) and ELISA (FIG. 31B).
FIG. 32A shows that the migratory response of cells is mediated in
part by osteopontin (Green=migrated cells (5.times.)). FIG. 32B
shows that neutralizing antibodies (NAb) to osteopontin reduce
renal cell migration response by 50%.
FIG. 33 shows that low-oxygen induction of cells modulates
expression of tissue remodeling genes.
FIG. 34 depicts a putative mechanism for low oxygen augmentation of
bioactivity of cells leading to renal regeneration.
FIG. 35 shows detection of microvesicles via a Western blot.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to heterogenous mixtures or
fractions of bioactive renal cells (BRCs) and methods of isolating
and culturing the same, as well as methods of treating a subject in
need with BRCs and/or BRC-containing constructs formed from a
scaffold seeded with BRCs as described herein. The bioactive renal
cells may be isolated renal cells including tubular and
erythropoietin (EPO)-producing kidney cells. The BRC cell
populations may include enriched tubular and EPO-producing cell
populations. The BRCs may be derived from or are themselves renal
cell fractions from healthy individuals. In addition, the present
invention provides renal cell fractions obtained from an unhealthy
individual may lack certain cellular components when compared to
the corresponding renal cell fractions of a healthy individual, yet
still retain therapeutic properties. The present invention also
provides therapeutically-active cell populations lacking cellular
components compared to a healthy individual, which cell populations
can be, in one embodiment, isolated and expanded from autologous
sources in various disease states.
The present invention also relates methods of providing a
regenerative effect to a native kidney by in vivo contacting the
native kidney with products secreted by renal cells, as well as
methods of preparing the secreted products. The present invention
further relates to the use of markers to determine the presence of
renal regeneration following treatment with a method described
herein.
Definitions
Unless defined otherwise, technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Principles of Tissue Engineering, 3.sup.rd Ed. (Edited by R Lanza,
R Langer, & J Vacanti), 2007 provides one skilled in the art
with a general guide to many of the terms used in the present
application. One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described.
The term "cell population" as used herein refers to a number of
cells obtained by isolation directly from a suitable tissue source,
usually from a mammal. The isolated cell population may be
subsequently cultured in vitro. Those of ordinary skill in the an
will appreciate that various methods for isolating and culturing
cell populations for use with the present invention and various
numbers of cells in a cell population that are suitable for use in
the present invention. A cell population may be an unfractionated,
heterogeneous cell population derived from the kidney. For example,
a heterogeneous cell population may be isolated from a kidney
biopsy or from whole kidney tissue. Alternatively, the
heterogeneous cell population may be derived from in vitro cultures
of mammalian cells, established from kidney biopsies or whole
kidney tissue. An unfractionated heterogeneous cell population may
also be referred to as a non-enriched cell population.
The term "native kidney" shall mean the kidney of a living subject.
The subject may be healthy or un-healthy. An unhealthy subject may
have a kidney disease.
The term "regenerative effect" shall mean an effect which provides
a benefit to a native kidney. The effect may include, without
limitation, a reduction in the degree of injury to a native kidney
or an improvement in, restoration of, or stabilization of a native
kidney function. Renal injury may be in the form of fibrosis,
inflammation, glomerular hypertrophy, etc. and related to kidney
disease in the subject.
The term "admixture" as used herein refers to a combination of two
or more isolated, enriched cell populations derived from an
unfractionated, heterogeneous cell population. According to certain
embodiments, the cell populations of the present invention are
renal cell populations.
An "enriched" cell population or preparation refers to a cell
population derived from a starting kidney cell population (e.g., an
unfractionated, heterogeneous cell population) that contains a
greater percentage of a specific cell type than the percentage of
that cell type in the starting population. For example, a starting
kidney cell population can be enriched for a first, a second, a
third, a fourth, a fifth, and so on, cell population of interest.
As used herein, the terror "cell population", "cell preparation"
and "cell prototype" are used interchangeably.
In one aspect, the term "enriched" cell population as used herein
refers to a cell population derived from a starting kidney cell
population (e.g., a cell suspension from a kidney biopsy or
cultured mammalian kidney cells) that contains a percentage of
cells capable of producing EPO that is greater than the percentage
of cells capable of producing EPO in the starting population. For
example, the term "B4" is a cell population derived from a starting
kidney cell population that contains a greater percentage of
EPO-producing cells, glomerular cells, and vascular cells as
compared to the starting population. The cell populations of the
present invention may be enriched for one or more cell types and
depleted of one or more other cell types. For example, an enriched
EPO-producing cell population may be enriched for interstitial
fibroblasts and depleted of tubular cells and collecting duct
epithelial cells relative to the interstitial fibroblasts and
tubular cells in a non-enriched cell population, i.e. the starting
cell population from which the enriched cell population is derived.
In all embodiments citing EPO-enriched or "B4" populations, the
enriched cell populations are heterogeneous populations of cells
containing cells that can produce EPO in an oxygen-regulated
manner, as demonstrated by oxygen-tunable EPO expression from the
endogenous native EPO gene.
In another aspect, an enriched cell population, which contains a
greater percentage of a specific cell type, e.g., vascular,
glomerular, or endocrine cells, than the percentage of that cell
type in the starting population, may also lack or be deficient in
one or more specific cell types, e.g., vascular, glomerular, or
endocrine cells, as compared to a starting kidney cell population
derived from a healthy individual or subject. For example, the term
"B4'," or B4 prime," in one aspect, is a cell population derived
from a starting kidney cell population that lacks or is deficient
in one or more cell types, e.g., vascular, glomerular or endocrine,
depending on the disease state of the starting specimen, as
compared to a healthy individual. In one embodiment, the B4' cell
population is derived from a subject having chronic kidney disease.
In one embodiment, the B4' cell population is derived from a
subject having focal segmental glomerulosclerosis (FSGS). In
another embodiment, the B4' cell population is derived from a
subject having autoimmune glomerulonephritis. In another aspect,
B4' is a cell population derived from a starting cell population
including all cell types, e.g., vascular, glomerular, or endocrine
cells, which is later depleted of or made deficient in one or more
cell types, e.g., vascular, glomerular, or endocrine cells. In yet
another aspect, B4' is a cell population derived from a starting
cell population including all cell types, e.g., vascular,
glomerular, or endocrine cells, in which one or more specific cell
types e.g., vascular, glomerular, or endocrine cells, is later
enriched. For example, in one embodiment, a B4' cell population may
be enriched for vascular cells but depleted of glomerular and/or
endocrine cells. In another embodiment, a B4' cell population may
be enriched for glomerular cells but depleted of vascular and/or
endocrine cells. In another embodiment, a B4' cell population may
be enriched for endocrine cells but depleted of vascular and/or
glomerular cells. In another embodiment, a B4' cell population may
be enriched for vascular and endocrine cells but depleted of
glomerular cells. In preferred embodiments, the B4' cell
population, alone or admixed with another enriched cell population,
e.g., B2 and/or B3, retains therapeutic properties. A B4' cell
population, for example, is described herein in the Examples, e.g.,
Examples 7-9.
In another aspect, an enriched cell population may also refer to a
cell population derived from a starting kidney cell population as
discussed above that contains a percentage of cells expressing one
or more tubular cell markers that is greater than the percentage of
cells expressing one or more tubular cell markers in the starting
population. For example, the term "B2" refers to a cell population
derived from a starting kidney cell population that contains a
greater percentage of tubular cells as compared to the starting
population. In addition, a cell population enriched for cells that
express one or more tubular cell markers (or "B2") may contain some
epithelial cells from the collecting duct system. Although the cell
population enriched for cells that express one or more tubular cell
markers (or "B2") is relatively depleted of EPO-producing cells,
glomerular cells, and vascular cells, the enriched population may
contain a smaller percentage of these cells (EPO-producing,
glomerular, and vascular) in comparison to the starting population.
In general, a heterogeneous cell population is depleted of one or
more cell types such that the depleted cell population contains a
lesser proportion of the cell type(s) relative to the proportion of
the cell type(s) contained in the heterogeneous cell population
prior to depletion. The cell types that may be depleted are any
type of kidney cell. For example, in certain embodiments, the cell
types that may be depleted include cells with large granularity of
the collecting duct and tubular system having a density of
<about 1.045 g/ml, referred to as "B1". In certain other
embodiments, the cell types that may be depleted include debris and
small cells of low granularity and viability having a density of
>about 1.095 g/ml, referred to as "B5". In some embodiments, the
cell population enriched for tubular cells is relatively depleted
of all of the following: "B1", "B5", oxygen-tunable EPO-expressing
cells, glomerular cells, and vascular cells.
The term "hypoxic" culture conditions as used herein refers to
culture conditions in which cells are subjected to a reduction in
available oxygen levels in the culture system relative to standard
culture conditions in which cells are cultured at atmospheric
oxygen levels (about 21%). Non-hypoxic conditions are referred to
herein as normal or normoxic culture conditions.
The term "oxygen-tunable" as used herein refers to the ability of
cells to modulate gene expression (up or down) based on the amount
of oxygen available to the cells. "Hypoxia-inducible" refers to the
upregulation of gene expression in response to a reduction in
oxygen tension (regardless of the pre-induction or starting oxygen
tension).
The term "biomaterial" as used here refers to a natural or
synthetic biocompatible material that is suitable for introduction
into living tissue. A natural biomaterial is a material that is
made by a living system. Synthetic biomaterials are materials which
are not made by a living system. The biomaterials disclosed herein
may be a combination of natural and synthetic biocompatible
materials. As used herein, biomaterials include, for example,
polymeric matrices and scaffolds. Those of ordinary skill in the
art will appreciate that the biomaterial(s) may be configured in
various forms, for example, as liquid hydrogel suspensions, porous
foam, and may comprise one or more natural or synthetic
biocompatible materials.
The term "anemia" as used herein refers to a deficit in red blood
cell number and/or hemoglobin levels due to inadequate production
of functional EPO protein by the EPO-producing cells of a subject,
and/or inadequate release of EPO protein into systemic circulation,
and/or the inability of erythroblasts in the bone marrow to respond
to EPO protein. A subject with anemia is unable to maintain
erythroid homeostasis. In general, anemia can occur with a decline
or loss of kidney function (e.g., chronic renal failure), anemia
associated with relative EPO deficiency, anemia associated with
congestive heart failure, anemia associated with myelo-suppressive
therapy such as chemotherapy or anti-viral therapy (e.g., AZT),
anemia associated with non-myeloid cancers, anemia associated with
viral infections such as HIV, and anemia of chronic diseases such
as autoimmune diseases (e.g., rheumatoid arthritis), liver disease,
and multi-organ system failure.
The term "EPO-deficiency" refers to any condition or disorder that
is treatable with an erythropoietin receptor agonist (e.g.,
recombinant EPO or EPO analogs), including anemia.
The term "kidney disease" as used herein refers to disorders
associated with any stage or degree of acute or chronic renal
failure that results in a loss of the kidney's ability to perform
the function of blood filtration and elimination of excess fluid,
electrolytes, and wastes from the blood. Kidney disease also
includes endocrine dysfunctions such as anemia
(erythropoietin-deficiency), and mineral imbalance (Vitamin D
deficiency). Kidney disease may originate in the kidney or may be
secondary to a variety of conditions, including (but not limited
to) heart failure, hypertension, diabetes, autoimmune disease, or
liver disease. Kidney disease may be a condition of chronic renal
failure that develops after an acute injury to the kidney. For
example, injury to the kidney by ischemia and/or exposure to
toxicants may cause acute renal failure incomplete recovery after
acute kidney injury may lead to the development of chronic renal
failure.
The term "treatment" refers to both therapeutic treatment and
prophylactic or preventative measures for kidney disease, anemia,
EPO deficiency, tubular transport deficiency, or glomerular
filtration deficiency wherein the object is to reverse, prevent or
slow down (lessen) the targeted disorder. Those in need of
treatment include those already having a kidney disease, anemia,
EPO deficiency, tubular transport deficiency, or glomerular
filtration deficiency as well as those prone to having a kidney
disease, anemia. EPO deficiency, tubular transport deficiency, or
glomerular filtration deficiency or those in whom the kidney
disease, anemia, EPO deficiency, tubular transport deficiency, or
glomerular filtration deficiency is to be prevented. The term
"treatment" as used herein includes the stabilization and/or
improvement of kidney function.
The term "in vivo contacting" as used herein refers to direct
contact in vivo between products secreted by an enriched population
of renal cells (or an admixture or construct containing renal
cells/renal cell fractions) and a native kidney. The direct in vivo
contacting may be paracrine, endocrine, or juxtacrine in nature.
The products secreted may be a heterogeneous population of
different products described herein.
The term "ribonucleic acid" or "RNA" as used herein refers to a
chain of nucleotide units where each unit is made up of a
nitrogenous base, a ribose sugar, and a phosphate. The RNA may be
in single or double stranded form. The RNA may be part of, within,
or associated with a vesicle. The vesicle may be an exosome. RNA
includes, without limitation, mRNAs, rRNA, small RNAs, snRNAs,
snoRNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), and
noncoding RNAs. The RNA is preferably human RNA.
The term "construct" refers to one or more cell populations
deposited on or in a surface of a scaffold or matrix made up of one
or more synthetic or naturally-occurring biocompatible materials.
The one or more cell populations may be coated with, deposited on,
embedded in, attached to, seeded, or entrapped in a biomaterial
made up of one or more synthetic or naturally-occurring
biocompatible polymers, proteins, or peptides. The one or more cell
populations may be combined with a biomaterial or scaffold or
matrix in vitro or in vivo. In general, the one or more
biocompatible materials used to form the scaffold/biomaterial is
selected to direct, facilitate, or permit the formation of
multicellular, three-dimensional, organization of at least one of
the cell populations deposited thereon. The one or more
biomaterials used to generate the construct may also be selected to
direct, facilitate, or permit dispersion and/or integration of the
construct or cellular components of the construct with the
endogenous host tissue, or to direct, facilitate, or permit the
survival, engraftment, tolerance, or functional performance of the
construct or cellular components of the construct.
The term "marker" or "biomarker" refers generally to a DNA, RNA,
protein, carbohydrate, or glycolipid-based molecular marker, the
expression or presence of which in a cultured cell population can
be detected by standard methods (or methods disclosed herein) and
is consistent with one or more cells in the cultured cell
population being a particular type of cell. The marker may be a
polypeptide expressed by the cell or an identifiable physical
location on a chromosome, such as a gene, a restriction
endonuclease recognition site or a nucleic acid encoding a
polypeptide (e.g., an mRNA) expressed by the native cell. The
marker may be an expressed region of a gene referred to as a "gene
expression marker", or some segment of DNA with no known coding
function. The biomarkers may be cell-derived, e.g., secreted,
products.
The terms "differentially expressed gene," "differential gene
expression" and their synonyms, which are used interchangeably,
refer to a gene whose expression is activated to a higher or lower
level in a first cell or cell population, relative to its
expression in a second cell or cell population. The terms also
include genes whose expression is activated to a higher or lower
level at different stages over time during passage of the first or
second cell in culture. It is also understood that a differentially
expressed gene may be either activated or inhibited at the nucleic
acid level or protein level, or may be subject to alternative
splicing to result in a different polypeptide product. Such
differences may be evidenced by a change in mRNA levels, surface
expression, secretion or other partitioning of a polypeptide, for
example. Differential gene expression may include a comparison of
expression between two or more genes or their gene products, or a
comparison of the ratios of the expression between two or more
genes or their gene products, or even a comparison of two
differently processed products of the same gene, which differ
between the first cell and the second cell. Differential expression
includes both quantitative, as well as qualitative, differences in
the temporal or cellular expression pattern in a gene or its
expression products among, for example, the first cell and the
second cell. For the purpose of this invention, "differential gene
expression" is considered to be present when there is a difference
between the expression of a given gene in the first cell and the
second cell. The differential expression of a marker may be in
cells from a patient before administration of a cell population,
admixture, or construct (the first cell) relative to expression in
cells from the patient after administration (the second cell).
The terms "inhibit", "down-regulate", "under-express" and "reduce"
are used interchangeably and mean that the expression of a gene, or
level of RNA molecules or equivalent RNA molecules encoding one or
more proteins or protein subunits, or activity of one or more
proteins or protein subunits, is reduced relative to one or more
controls, such as, for example, one or more positive and/or
negative controls. The under-expression may be in cells from a
patient before administration of a cell population, admixture, or
construct relative to cells from the patient after
administration.
The term "up-regulate" or "over-express" is used to mean that the
expression of a gene, or level of RNA molecules or equivalent RNA
molecules encoding one or more proteins or protein subunits, or
activity of one or more proteins or protein subunits, is elevated
relative to one or more controls, such as, for example, one or more
positive and/or negative controls. The over-expression may be in
cells from a patient after administration of a cell population,
admixture, or construct relative to cells from the patient before
administration.
The term "subject" shall mean any single human subject, including a
patient, eligible for treatment, who is experiencing or has
experienced one or more signs, symptoms, or other indicators of a
kidney disease, anemia, or EPO deficiency. Such subjects include
without limitation subjects who are newly diagnosed or previously
diagnosed and are now experiencing a recurrence or relapse, or are
at risk for a kidney disease, anemia, or EPO deficiency, no matter
the cause. The subject may have been previously treated for a
kidney disease, anemia, or EPO deficiency, or not so treated.
The term "patient" refers to any single animal, more preferably a
mammal (including such non-human animals as, for example, dogs,
cats, horses, rabbits, zoo animals, cows, pigs, sheep, and
non-human primates) for which treatment is desired. Most
preferably, the patient herein is a human.
The term "sample" or "patient sample" or "biological sample" shall
generally mean any biological sample obtained from a subject or
patient, body fluid, body tissue, cell line, tissue culture, or
other source. The term includes tissue biopsies such as, for
example, kidney biopsies. The term includes cultured cells such as,
for example, cultured mammalian kidney cells. Methods for obtaining
tissue biopsies and cultured cells from mammals are well known in
the art. If the term "sample" is used alone, it shall still mean
that the "sample" is a "biological sample" or "patient sample",
i.e., the terms are used interchangeably.
The term "test sample" refers to a sample from a subject that has
been treated by a method of the present invention. The test sample
may originate from various sources in the mammalian subject
including, without limitation, blood, semen, serum, urine, hone
marrow, mucosa, tissue, etc.
The term "control" or "control sample" refers a negative or
positive control in which a negative or positive result is expected
to help correlate a result in the test sample. Controls that are
suitable for the present invention include, without limitation, a
sample known to exhibit indicators characteristic of normal
erythroid homeostasis, a sample known to exhibit indicators
characteristic of anemia, a sample obtained from a subject known
not to be anemic, and a sample obtained from a subject known to be
anemic. Additional controls suitable for use in the methods of the
present invention include, without limitation, samples derived from
subjects that have been treated with pharmacological agents known
to modulate erythropoiesis (e.g., recombinant EPO or EPO analogs).
In addition, the control may be a sample obtained from a subject
prior to being treated by a method of the present invention. An
additional suitable control may be a test sample obtained from a
subject known to have any type or stage of kidney disease, and a
sample from a subject known not to have any type or stage of kidney
disease. A control may be a normal healthy matched control. Those
of skill in the art will appreciate other controls suitable for use
in the present invention.
"Regeneration prognosis", "regenerative prognosis", or "prognostic
for regeneration" generally refers to a forecast or prediction of
the probable regenerative course or outcome of the administration
or implantation of a cell population, admixture or construct
described herein. For a regeneration prognosis, the forecast or
prediction may be informed by one or more of the following:
improvement of a functional kidney after implantation or
administration, development of a functional kidney after
implantation or administration, development of improved kidney
function or capacity after implantation or administration, and
expression of certain markers by the native kidney following
implantation or administration.
"Regenerated kidney" refers to a native kidney after implantation
or administration of a cell population, admixture, or construct as
described herein. The regenerated kidney is characterized by
various indicators including, without limitation, development of
function or capacity in the native kidney, improvement of function
or capacity in the native kidney, and the expression of certain
markers in the native kidney. Those of ordinary skill in the art
will appreciate that other indicators may be suitable for
characterizing a regenerated kidney.
Cell Populations
Isolated, heterogeneous populations of kidney cells, and admixtures
thereof, enriched for specific bioactive components or cell types
and/or depleted of specific inactive or undesired components or
cell types for use in the treatment of kidney disease, i.e.,
providing stabilization and/or improvement and/or regeneration of
kidney function, were previously described in U.S. application Ser.
No. 12/617,721 filed Nov. 12, 2009, the entire contents of which is
incorporated herein by reference. The present invention provides
isolated renal cell fractions that lack cellular components as
compared to a healthy individual yet retain therapeutic properties,
i.e., provide stabilization and/or improvement and/or regeneration
of kidney function. The cell populations, cell fractions, and/or
admixtures of cells described herein may be derived from healthy
individuals, individuals with a kidney disease, or subjects as
described herein.
Bioactive Cell Populations
In one aspect, the present invention is based on the surprising
finding that certain subtractions of a heterogeneous population of
renal cells, enriched for bioactive components and depleted of
inactive or undesired components, provide superior therapeutic and
regenerative outcomes than the starting population. For example,
bioactive components of the invention, e.g., B2, B4, and B3, which
are depleted of inactive or undesired components, e.g., B1 and B5,
alone or admixed, provide unexpected stabilization and/or
improvement and/or regeneration of kidney function.
In another aspect, the present invention is based on the surprising
finding that a specific subtraction, B4, depleted of or deficient
in one or more cell types, e.g., vascular, endocrine, or
endothelial, i.e., B4', retains therapeutic properties, e.g.,
stabilization and/or improvement and/or regeneration of kidney
function, alone or when admixed with other bioactive subfractions,
e.g., B2 and/or B3. In a preferred embodiment, the bioactive cell
population is B2. In certain embodiments, the B2 cell population is
admixed with B4 or B4'. In other embodiments, the B2 cell
population is admixed with B3. In other embodiments, the B2 cell
population is admixed with both B3 and B4, or specific cellular
components of B3 and/or B4.
The B2 cell population is characterized by expression of a tubular
cell marker selected from the group consisting of one or more of
the following: megalin, cubilin, hyaluronic acid synthase 2 (HAS2),
Vitamin D3 25-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin
(Ecad), Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS
oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD
domain-containing ion transport regulator 4 (Fxyd4), solute carrier
family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde
dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase
1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8), and
collecting duct marker Aquaporin-4 (Aqp4). B2 is larger and more
granulated than B3 and/or B4 and thus having a buoyant density
between about 1.045 g/ml and about 1.063 g/ml (rodent), between
about 1.045 g/ml and 1.052 g/ml (human), and between about 1.045
g/ml and about 1.058 g/ml (canine).
The B3 cell population is characterized by the expression of
vascular, glomerular and proximal tubular markers with some
EPO-producing cells, being of an intermediate size and granularity
in comparison to B2 and B4, and thus having a buoyant density
between about 1.063 g/ml and about 1.073 g/ml (rodent), between
about 1.052 g/ml and about 1.063 g/ml (human), and between about
1.058 g/ml and about 1.063 g/ml (canine). B3 is characterized by
expression of markers selected from the group consisting of one or
more of the following: aquaporin 7 (Aqp7), FXYD domain-containing
ion transport regulator 2 (Fxyd2), solute carrier family 17 (sodium
phosphate), member 3 (Slc17a3), solute carrier family 3, member 1
(Slc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase (Napsa),
solute carrier family 2 (facilitated glucose transporter), member 2
(Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane
protein 27 (Tmem27), acyl-CoA synthetase medium-chain family member
2 (Acsm2), glutathione peroxidase 3 (Gpx3),
fructose-1,6-biphosphatase 1 (Fbp1), and alanine-glyoxylate
aminotransferase 2 (Agxt2). B3 is also characterized by the
vascular expression marker Platelet endothelial cell adhesion
molecule (Pecam) and the glomerular expression marker podocin
(Podn).
The B4 cell population is characterized by the expression of a
vascular marker set containing one or more of the following: PECAM,
VEGF, KDR, HIF1a, CD31, CD146, a glomerular marker set containing
one or more of the following: Podocin (Podn), and Nephrin (Neph);
and an oxygen-turnable EPO enriched population compared to
unfractionated (UNFX), B2 and B3. B4 is also characterized by the
expression of one or more of the following markers: chemokine
(C--X--C motif) receptor 4 (Cxcr4), endothelin receptor type B
(Ednrb), collagen, type V, alpha 2 (Col5a2). Cadherin 5 (Cdh5),
plasminogen activator, tissue (Plat), angiopoietin 2 (Angpt2),
kinase insert domain protein receptor (Kdr), secreted protein,
acidic, cysteine-rich (osteonectin) (Sparc), serglycin (Srgn), TIMP
metallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1),
wingless-type MMTV integration site family, member 4 (Wnt4),
regulator of G-protein signaling 4 (Rgs4), Platelet endothelial
cell adhesion molecule (Pecam), and Erythropoietin (Epo). B4 is
also characterized by smaller, less granulated cells compared to
either B2 or B3, with a buoyant density between about 1.073 g/ml
and about 1.091 g/ml (rodent), between about 1.063 g/ml and about
1.091 g/mL (human and canine).
The B4' cell population is defined as having a buoyant density of
between 1.063 g/mL and 1.091 g/mL and expressing one or more of the
following markers: PECAM, vEGF, KDR, HIF1a, podocin, nephrin, EPO,
CK7, CK8/18/19. In one embodiment, the B4' cell population is
characterized by the expression of a vascular marker set containing
one or more of the following: PECAM, vEGF, KDR, HIF1a, CD31, CD146.
In another embodiment, the B4' cell population is characterized by
the expression of an endocrine marker EPO. In one embodiment, the
B4' cell population is characterized by the expression of a
glomerular marker set containing one or more of the following:
Podocin (Podn), and Nephrin (Neph). In certain embodiments, the B4'
cell population is characterized by the expression of a vascular
marker set containing one or more of the following: PECAM, vEGF,
KDR, HIF1a and by the expression of an endocrine marker EPO. In
another embodiment, B4' is also characterized by smaller, less
granulated cells compared to either B2 or B3, with a buoyant
density between about 1.073 g/ml and about 1.091 g/ml (rodent),
between about 1.063 g/ml and about 1.091 g/mL (human and
canine).
In one aspect, the present invention provides an isolated, enriched
B4' population of human renal cells comprising at least one of
erythropoietin (EPO)-producing cells, vascular cells, and
glomerular cells having a density between 1.063 g/mL and 1.091
g/mL. In one embodiment, the B4' cell population is characterized
by expression of a vascular marker. In certain embodiments, the B4'
cell population is not characterized by expression of a glomerular
marker. In some embodiments, the B4' cell population is capable of
oxygen-tunable erythropoietin (EPO) expression.
In one embodiment, the B4' cell population does not include a B2
cell population comprising tubular cells having a density between
1.045 g/mL and 1.052 g/mL. In another embodiment, the B4' cell
population does not include a B1 cell population comprising large
granular cells of the collecting duct and tubular system having a
density of <1.045 g/ml. In yet another embodiment, the B4' cell
population does not include a B5 cell population comprising debris
and small cells of low granularity and viability with a
density>1.091 g/ml.
In one embodiment, the B4' cell population does not include a B2
cell population comprising tubular cells having a density between
1.045 g/mL and 1.052 g/mL; a B1 cell population comprising large
granular cells of the collecting duct and tubular system having a
density of <1.045 g/ml; and a B5 cell population comprising
debris and small cells of low granularity and viability with a
density>1.091 g/ml. In some embodiments, the B4' cell population
may be derived from a subject having kidney disease.
In one aspect, the present invention provides an admixture of human
renal cells comprising a first cell population, B2, comprising an
isolated, enriched population of tubular cells having a density
between 1.045 g/mL and 1.052 g/mL, and a second cell population,
B4', comprising erythropoietin (EPO)-producing cells and vascular
cells but depleted of glomerular cells having a density between
about 1.063 g/mL and 1.091 g/mL, wherein the admixture does not
include a B1 cell population comprising large granular cells of the
collecting duct and tubular system having a density of <1.045
g/ml, or a B5 cell population comprising debris and small cells of
low granularity and viability with a density>1.091 g/ml. In
certain embodiment, the B4' cell population is characterized by
expression of a vascular marker. In one embodiment, the B4' cell
population is not characterized by expression of a glomerular
marker. In certain embodiments, B2 limiter comprises collecting
duct epithelial cells. In one embodiment, the admixture of cells is
capable of receptor-mediated albumin uptake. In another embodiment,
the admixture of cells is capable of oxygen-tunable erythropoietin
(EPO) expression. In one embodiment, the admixture contains
HAS-2-expressing cells capable of producing and/or stimulating the
production of high-molecular weight species of hyaluronic acid (HA)
both in vitro and in vivo. In all embodiments, the first and second
cell populations may be derived from kidney tissue or cultured
kidney cells.
In one embodiment, the admixture is capable of providing a
regenerative stimulus upon in vivo delivery. In other embodiments,
the admixture is capable of reducing the decline of, stabilizing,
or improving glomerular filtration, tubular resorption, urine
production, and/or endocrine function upon in vivo delivery. In one
embodiment, the B4' cell population is derived from a subject
having kidney disease.
In one aspect, the present invention provides an isolated, enriched
B4' population of human renal cells comprising at least one of
erythropoietin (EPO)-producing cells, vascular cells, and
glomerular cells having a density between 1.063 g/mL and 1.091
g/mL. In one embodiment, the B4' cell population is characterized
by expression of a vascular marker. In certain embodiments, the B4'
cell population is not characterized by expression of a glomerular
marker. The glomerular marker that is not expressed may be podocin
(see Example 7). In some embodiments, the B4' cell population is
capable of oxygen-tunable erythropoietin (EPO) expression.
In one embodiment, the B4' cell population does not include a B2
cell population comprising tubular cells having a density between
1.045 g/mL and 1.052 g/mL. In another embodiment, the B4' cell
population does not include a B1 cell population comprising large
granular cells of the collecting duct and tubular system having a
density of <1.045 g/ml. In yet another embodiment, the B4' cell
population does not include a B5 cell population comprising debris
and small cells of low granularity and viability with a
density>1.091 g/ml.
In one embodiment, the B4' cell population does not include a B2
cell population comprising tubular cells having a density between
1.045 g/mL and 1.052 g/mL; a B1 cell population comprising large
granular cells of the collecting duct and tubular system having a
density of <1.045 g/ml; and a B5 cell population comprising
debris and small cells of low granularity and viability with a
density>1.091 g/ml.
In some embodiments, the B4' cell population may be derived from a
subject having kidney disease. In one aspect, the present invention
provides an admixture of human renal cells comprising a first cell
population, B2, comprising an isolated, enriched population of
tubular cells having a density between 1.045 g/mL and 1.052 g/mL,
and a second cell population, B4', comprising erythropoietin
(EPO)-producing cells and vascular cells but depleted of glomerular
cells having a density between about 1.063 g/mL and 1.091 g/mL,
wherein the admixture does not include a B1 cell population
comprising large granular cells of the collecting duct and tubular
system having a density of <1.045 g/ml, or a B5 cell population
comprising debris and small cells of low granularity and viability
with a density>1.091 g/ml. In certain embodiment, the B4' cell
population is characterized by expression of a vascular marker. In
one embodiment, the B4' cell population is not characterized by
expression of a glomerular marker. In certain embodiments, B2
further comprises collecting duct epithelial cells. In one
embodiment, the admixture of cells is capable of receptor-mediated
albumin uptake. In another embodiment, the admixture of cells is
capable of oxygen-tunable erythropoietin (EPO) expression. In one
embodiment, the admixture contains HAS-2-expressing cells capable
of producing and/or stimulating the production of high-molecular
weight species of hyaluronic acid (HA) both in vitro and in vivo.
In all embodiments, the first and second cell populations may be
derived from kidney tissue or cultured kidney cells.
In one embodiment, the admixture is capable of providing a
regenerative stimulus upon in vivo delivery. In other embodiments,
the admixture is capable of reducing the decline of, stabilizing,
or improving glomerular filtration, tubular resorption, urine
production, and/or endocrine function upon in vivo delivery. In one
embodiment, the B4' cell population is derived from a subject
having kidney disease.
In a preferred embodiment, the admixture comprises B2 in
combination with B3 and/or B4. In another preferred embodiment, the
admixture comprises B2 in combination with B3 and/or B4'. In other
preferred embodiments, the admixture consists of or consists
essentially of (i) B2 in combination with B3 and/or B4; or (ii) B2
in combination with B3 and/or B4'.
The admixtures that contain a B4' cell population may contain B2
and/or B3 cell populations that are also obtained from a
non-healthy subject. The non-healthy subject may be the same
subject from which the B4' fraction was obtained. In contrast to
the B4' cell population, the B2 and B3 cell populations obtained
from non-healthy subjects are typically not deficient in one or
more specific cell types as compared to a starting kidney cell
population derived from a healthy individual.
Hyaluronic Acid Production by B2 and B4
Hyaluronan (also called hyaluronic acid or hyaluronate) is a
glycosaminoglycan (GAG), which consists of a regular repeating
sequence of non-sulfated disaccharide units, specifically
N-acetylglucosamine and glucuronic acid. Its molecular weight can
range from 400 daltons (the disaccharide) to over a million
daltons. It is found in variable amounts in all tissues, such as
the skin, cartilage, and eye, and in most if not all fluids in
adult animals. It is especially abundant in early embryos. Space
created by hyaluronan, and indeed GAGs in general, permit it to
play a role in cell migration, cell attachment, during wound
repair, organogenesis, immune cell adhesion, activation of
intracellular signalling, as well as tumour metastasis. These roles
are mediated by specific protein and proteoglycan binding to
Hyaluronan. Cell motility and immune cell adhesion is mediated by
the cell surface receptor RHAMM (Receptor for Hyaluronan-Mediated
Motility; Hardwick et al., 1992) and CD44 (Jalkenan et al., 1987;
Miyake et al., 1990). Hyaluronan is synthesized directly at the
inner membrane of the cell surface with the growing polymer
extruded through the membrane to the outside of the cell as it is
being synthesized. Synthesis is mediated by a single protein
enzyme, hyaluronan synthetase (HAS) whose gene family consists of
at least 3 members.
It has recently been shown that hyaluronic acid interacts with
CD44, and such interactions may, among other actions, recruit
non-resident cells (such as mesenchymal stem cells (MSCs)) to
injured renal tissue and enhance renal regeneration (Kidney
International (2007) 72, 430-441).
Unexpectedly, it has been found that the B2 and B4 cell
preparations are capable of expressing higher molecular weight
species of hyaluronic acid (HA) both in vitro and in vivo, through
the actions of hyaluronic acid synthase-2 (HAS-2)--a marker that is
enriched more specifically in the B2 cell population. Treatment
with B2 in a 5/6 Nx model was shown to reduce fibrosis, concomitant
with strong expression HAS-2 expression in vivo and the expected
production of high-molecular-weight HA within the treated tissue.
Notably, the 5/6 Nx model left untreated resulted in fibrosis with
limited detection of HAS-2 and little production of
high-molecular-weight HA. Without wishing to be bound by theory, it
is hypothesized that this anti-inflammatory high-molecular weight
species of HA produced predominantly by B2 (and to some degree by
B4) acts synergystically with the cell preparations in the
reduction of renal fibrosis and in the aid of renal regeneration.
Accordingly, the instant invention includes delivery of the
cellular prototypes of the invention in a biomaterial comprising
hyaluronic acid. Also contemplated by the instant invention is the
provision of a biomaterial component of the regenerative stimulus
via direct production or stimulation of production by the implanted
cells.
In one aspect, the present invention provides isolated,
heterogeneous populations of EPO-producing kidney cells for use in
the treatment of kidney disease, anemia and/or EPO deficiency in a
subject in need. In one embodiment, the cell populations are
derived from a kidney biopsy. In another embodiment, the cell
populations are derived from whole kidney tissue. In one other
embodiment, the cell populations are derived from in vitro cultures
of mammalian kidney cells, established from kidney biopsies or
whole kidney tissue. In all embodiments, these populations are
unfractionated cell populations, also referred to herein as
non-enriched cell populations.
In another aspect, the present invention provides isolated
populations of erythropoietin (EPO)-producing kidney cells that are
further enriched such that the proportion of EPO-producing cells in
the enriched subpopulation is greater relative to the proportion of
EPO-producing cells in the starting or initial cell population. In
one embodiment, the enriched EPO-producing cell fraction contains a
greater proportion of interstitial fibroblasts and a lesser
proportion of tubular cells relative to the interstitial
fibroblasts and tubular cells contained in the unenriched initial
population. In certain embodiments, the enriched EPO-producing cell
fraction contains a greater proportion of glomerular cells and
vascular cells and a lesser proportion of collecting duct cells
relative to the glomerular cells, vascular cells and collecting
duct cells contained in the unenriched initial population. In such
embodiments, these populations are referred to herein as the "B4"
cell population.
In another aspect, the present invention provides an EPO-producing
kidney cell population that is admixed with one or more additional
kidney cell populations. In one embodiment, the EPO-producing cell
population is a first cell population enriched for EPO-producing
cells, e.g., B4. In another embodiment, the EPO-producing cell
population is a first cell population that is not enriched for
EPO-producing cells, e.g., B2. In another embodiment, the first
cell population is admixed with a second kidney cell population. In
some embodiments, the second cell population is enriched for
tubular cells, which may be demonstrated by the presence of a
tubular cell phenotype. In another embodiment, the tubular cell
phenotype may be indicated by the presence of one tubular cell
marker. In another embodiment, the tubular cell phenotype may be
indicated by the presence of one or more tubular cell markers. The
tubular cell markers include, without limitation, megalin, cubilin,
hyaluronic acid synthase 2 (HAS2), Vitamin D3 25-Hydroxylase
(CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), Aquaporin-1
(Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogene family
(Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion
transport regulator 4 (Fxyd4), solute carrier family 9
(sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde
dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase
1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8). In another
embodiment, the first cell population is admixed with at least one
of several types of kidney cells including, without limitation,
interstitium-derived cells, tubular cells, collecting duct-derived
cells, glomerulus-derived cells, and/or cells derived from the
blood or vasculature. The EPO-producing kidney cell population may
contain B4 or B4' in the form of an admixture with B2 and/or B3, or
in the form of an enriched cell population, e.g., B2+B3+B4/B4'.
In one aspect, the EPO-producing kidney cell populations of the
present invention are characterized by EPO expression and
bioresponsiveness to oxygen, such that a reduction in the oxygen
tension of the culture system results in an induction in the
expression of EPO. In one embodiment, the EPO-producing cell
populations are enriched for EPO-producing cells. In one
embodiment, the EPO expression is induced when the cell population
is cultured under conditions where the cells are subjected to a
reduction in available oxygen levels in the culture system as
compared to a cell population cultured at normal atmospheric
(.about.21%) levels of available oxygen. In one embodiment,
EPO-producing cells cultured in lower oxygen conditions express
greater levels of EPO relative to EPO-producing cells cultured at
normal oxygen conditions. In general, the culturing of cells at
reduced levels of available oxygen (also referred to as hypoxic
culture conditions) means that the level of reduced oxygen is
reduced relative to the culturing of cells at normal atmospheric
levels of available oxygen (also referred to as normal or normoxic
culture conditions). In one embodiment, hypoxic cell culture
conditions include culturing cells at about less than 1% oxygen,
about less than 2% oxygen, about less than 3% oxygen, about less
than 4% oxygen, or about less than 5% oxygen. In another
embodiment, normal or normoxic culture conditions include culturing
cells at about 10% oxygen, about 12% oxygen, about 13% oxygen,
about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17%
oxygen, about 18% oxygen, about 19% oxygen, about 20% oxygen, or
about 21% oxygen.
In one other embodiment, the induction or increased expression of
EPO is obtained and can be observed by culturing cells at about
less than 5% available oxygen and comparing EPO expression levels
to cells cultured at atmospheric (about 21%) oxygen. In another
embodiment, the induction of EPO is obtained in a culture of cells
capable of expressing EPO by a method that includes a first culture
phase in which the culture of cells is cultivated at atmospheric
oxygen (about 21%) for some period of time and a second culture
phase in which the available oxygen levels are reduced and the same
cells are cultured at about less than 5% available oxygen. In
another embodiment, the EPO expression that is responsive to
hypoxic conditions is regulated by HIF1.alpha.. Those of ordinary
skill in the all will appreciate that other oxygen manipulation
culture conditions known in the art may be used for the cells
described herein.
In one aspect, the enriched populations of EPO-producing mammalian
cells are characterized by bio-responsiveness (e.g., EPO
expression) to perfusion conditions. In one embodiment, the
perfusion conditions include transient, intermittent, or continuous
fluid flow (perfusion). In one embodiment, the EPO expression is
mechanically-induced when the media in which the cells are cultured
is intermittently or continuously circulated or agitated in such a
manner that dynamic forces are transferred to the cells via the
flow. In one embodiment, the cells subjected to the transient,
intermittent, or continuous fluid flow are cultured in such a
manner that they are present as three-dimensional structures in or
on a material that provides framework and/or space for such
three-dimensional structures to form. In one embodiment, the cells
are cultured on porous beads and subjected to intermittent or
continuous fluid flow by means of a rocking platform, orbiting
platform, or spinner flask. In another embodiment, the cells are
cultured on three-dimensional scaffolding and placed into a device
whereby the scaffold is stationary and fluid flows directionally
through or across the scaffolding. Those of ordinary skill in the
art will appreciate that other perfusion culture conditions known
in the art may be used for the cells described herein.
Inactive Cell Populations
As described herein, the present invention is based, in part, on
the surprising finding that certain subtractions of a heterogeneous
population of renal cells, enriched for bioactive components and
depleted of inactive or undesired components, provide superior
therapeutic and regenerative outcomes than the starting population.
In preferred embodiments, the cellular populations of the instant
invention are depleted of B1, and/or B5 cell populations. For
instance, the following may be depleted of B1 and/or B5: admixtures
of two or more of B2, B3, and B4'; an enriched cell population of
B2, B3, and B4'.
The B1 cell population comprises large, granular cells of the
collecting duct and tubular system, with the cells of the
population having a buoyant density less than about 1.045 g/m. The
B5 cell population is comprised of debris and small cells of low
granularity and viability and having a buoyant density greater than
about 1.0091 g/ml.
Methods of Isolating and Culturing Cell Populations
The present invention, in one aspect, provides methods for
separating and isolating renal cellular components, e.g., enriched
cell populations, for therapeutic use, including the treatment of
kidney disease, anemia, EPO deficiency, tubular transport
deficiency, and glomerular filtration deficiency. In one
embodiment, the cell populations are isolated from freshly
digested, i.e., mechanically or enzymatically digested, kidney
tissue or from heterogeneous in vitro cultures of mammalian kidney
cells.
It has unexpectedly been discovered that culturing heterogeneous
mixtures of renal cells in hypoxic culture conditions prior to
separation on a density gradient provides for enhanced distribution
and composition of cells in both B4, including B4', and B2 and/or
B3 fractions. The enrichment of oxygen-dependent cells in B4 from
B2 was observed for renal cells isolated from both diseased and
non-diseased kidneys. Without wishing to be bound by theory, this
may be due to one or more of the following phenomena: 1) selective
survival, death, or proliferation of specific cellular components
during the hypoxic culture period; 2) alterations in cell
granularity and/or size in response to the hypoxic culture, thereby
effecting alterations in buoyant density and subsequent
localization during density gradient separation; and 3) alterations
in cell gene/protein expression in response to the hypoxic culture
period, thereby resulting in differential characteristics of the
cells within any given fraction of the gradient. Thus, in one
embodiment, the cell populations enriched for tubular cells, e.g.,
B2, are hypoxia-resistant.
Exemplary techniques for separating and isolating the cell
populations of the invention include separation on a density
gradient based on the differential specific gravity of different
cell types contained within the population of interest. The
specific gravity of any given cell type can be influenced by the
degree of granularity within the cells, the intracellular volume of
water, and other factors. In one aspect, the present invention
provides optimal gradient conditions for isolation of the cell
preparations of the instant invention, e.g., B2 and B4, including
B4', across multiple species including, but not limited to, human,
canine, and rodent. In a preferred embodiment, a density gradient
is used to obtain a novel enriched population of tubular cells
fraction, i.e., B2 cell population, derived from a heterogeneous
population of renal cells. In one embodiment, a density gradient is
used to obtain a novel enriched population of EPO-producing cells
fraction, i.e., B4 cell population, derived from a heterogeneous
population of renal cells. In other embodiments, a density gradient
is used to obtain enriched subpopulations of tubular cells,
glomerular cells, and endothelial cells of the kidney. In one
embodiment, both the EPO-producing and the tubular cells are
separated from the red blood cells and cellular debris. In one
embodiment, the EPO-producing, glomerular, and vascular cells are
separated from other cell types and from red blood cells and
cellular debris, while a subpopulation of tubular cells and
collecting duct cells are concomitantly separated from other cell
types and from red blood cells and cellular debris. In one other
embodiment, the endocrine, glomerular, and/or vascular cells are
separated from other cell types and from red blood cells and
cellular debris, while a subpopulation of tubular cells and
collecting duct cells are concomitantly separated from other cell
types and from red blood cells and cellular debris.
The instant invention generated the novel cell populations by
using, in part, the OPTIPREP.RTM. (Axis-Shield) density gradient
medium, comprising 60% nonionic iodinated compound iodixanol in
water, based on certain key features described below. One of skill
in the art, however, will recognize that any density gradient or
other means, e.g., immunological separation using cell surface
markers known in the art, comprising necessary features for
isolating the cell populations of the instant invention may be used
in accordance with the invention. It should also be recognized by
one skilled in the art that the same cellular features that
contribute to separation of cellular subpopulations via density
gradients (size and granularity) can be exploited to separate
cellular subpopulations via flow cytometry (forward scatter=a
reflection of size via flow cytometry, and side scatter=a
reflection of granularity). Importantly, the density gradient
medium should have low toxicity towards the specific cells of
interest. While the density gradient medium should have low
toxicity toward the specific cells of interest, the instant
invention contemplates the use of gradient mediums which play a
role in the selection process of the cells of interest. Without
wishing to be bound by theory, it appears that the cell populations
of the instant invention recovered by the gradient comprising
iodixanol are iodixanol-resistant, as there is an appreciable loss
of cells between the loading and recovery steps, suggesting that
exposure to iodixanol under the conditions of the gradient leads to
elimination of certain cells. The cells appearing in the specific
bands after the iodixanol gradient are resistant to any untoward
effects of iodixanol and/or density gradient exposure. Accordingly,
the present invention also contemplates the use of additional
contrast medias which are mild to moderate nephrotoxins in the
isolation and/or selection of the cell populations of the instant
invention. In addition, the density gradient medium should also not
bind to proteins in human plasma or adversely affect key functions
of the cells of interest.
In another aspect, the present invention provides methods of
enriching and/or depleting kidney cell types using fluorescent
activated cell sorting (FACS). In one embodiment, kidney cell types
may be enriched and/or depleted using BD FACSAria.TM. or
equivalent.
In another aspect, the present invention provides methods of
enriching and/or depleting kidney cell types using magnetic cell
sorting. In one embodiment, kidney cell types may be enriched
and/or depleted using the Miltenyi autoMACS.RTM. system or
equivalent.
In another aspect, the present invention provides methods of
three-dimensional culturing of the renal cell populations. In one
aspect, the present invention provides methods of culturing the
cell populations via continuous perfusion. In one embodiment, the
cell populations cultured via three-dimensional culturing and
continuous perfusion demonstrate greater cellularity and
interconnectivity when compared to cell populations cultured
statically. In another embodiment, the cell populations cultured
via three dimensional culturing and continuous perfusion
demonstrate greater expression of EPO, as well as enhanced
expression of renal tubule-associate genes such as e-cadherin when
compared to static cultures of such cell populations.
In yet another embodiment, the cell populations cultured via
continuous perfusion demonstrate greater levels of glucose and
glutamine consumption when compared to cell populations cultured
statically.
As described herein (including Example 3), low or hypoxic oxygen
conditions may be used in the methods to prepare the cell
populations of the present invention. However, the methods of the
present invention may be used without the step of low oxygen
conditioning. In one embodiment, normoxic conditions may be
used.
Those of ordinary skill in the art will appreciate that other
methods of isolation and culturing known in the art may be used for
the cells described herein.
Biomaterials (Polymeric Matrices or Scaffolds)
As described in Bertram et al. U.S. Published Application
20070276507 (incorporated herein by reference in its entirety),
polymeric matrices or scaffolds may be shaped into any number of
desirable configurations to satisfy any number of overall system,
geometry or space restrictions. In one embodiment, the matrices or
scaffolds of the present invention may be three-dimensional and
shaped to conform to the dimensions and shapes of an organ or
tissue structure. For example, in the use of the polymeric scaffold
for treating kidney disease, anemia, EPO deficiency, tubular
transport deficiency, or glomerular filtration deficiency, a
three-dimensional (3-D) matrix may be used. A variety of
differently shaped 3-D scaffolds may be used. Naturally, the
polymeric matrix may be shaped in different sizes and shapes to
conform to differently sized patients. The polymeric matrix may
also be shaped in other ways to accommodate the special needs of
the patient. In another embodiment, the polymeric matrix or
scaffold may be a biocompatible, porous polymeric scaffold. The
scaffolds may be formed from a variety of synthetic or
naturally-occurring materials including, but not limited to,
open-cell polylactic acid (OPLA.RTM.), cellulose ether, cellulose,
cellulosic ester, fluorinated polyethylene, phenolic,
poly-4-methylpentene, polyacrylonitrile, polyamide, polyamideimide,
polyacrylate, polybenzoxazole, polycarbonate, polycyanoarylether,
polyester, polyestercarbonate, polyether, polyetheretherketone,
polyetherimide, polyetherketone, polyethersulfone, polyethylene,
polyfluoroolefin, polyimide, polyolefin, polyoxadiazole,
polyphenylene oxide, polyphenylene sulfide, polypropylene,
polystyrene, polysulfide, polysulfone, polytetrafluoroethylene,
polythioether, polytriazole, polyurethane, polyvinyl,
polyvinylidene fluoride, regenerated cellulose, silicone,
urea-formaldehyde, collagens, laminins, fibronectin, silk, elastin,
alginate, hyaluronic acid, agarose, or copolymers or physical
blends thereof. Scaffolding configurations may range from liquid
hydrogel suspensions to soft porous scaffolds to rigid,
shape-holding porous scaffolds.
Hydrogels may be formed from a variety of polymeric materials and
are useful in a variety of biomedical applications. Hydrogels can
be described physically as three-dimensional networks of
hydrophilic polymers. Depending on the type of hydrogel, they
contain varying percentages of water, but altogether do not
dissolve in water. Despite their high water content, hydrogels are
capable of additionally binding great volumes of liquid due to the
presence of hydrophilic residues. Hydrogels swell extensively
without changing their gelatinous structure. The basic physical
features of hydrogel can be specifically modified, according to the
properties of the polymers used and the additional special
equipments of the products.
Preferably, the hydrogel is made of a polymer, a biologically
derived material, a synthetically derived material or combinations
thereof, that is biologically inert and physiologically compatible
with mammalian tissues. The hydrogel material preferably does not
induce an inflammatory response. Examples of other materials which
can be used to form a hydrogel include (a) modified alginates, (b)
polysaccharides (e.g. gellan gum and carrageenans) which gel by
exposure to monovalent cations, (c) polysaccharides (e.g.,
hyaluronic acid) that are very viscous liquids or are thixotropic
and form a gel over time by the slow evolution of structure, and
(d) polymeric hydrogel precursors (e.g., polyethylene
oxide-polypropylene glycol block copolymers and proteins). U.S.
Pat. No. 6,224,893 B1 provides a detailed description of the
various polymers, and the chemical properties of such polymers,
that are suitable for making hydrogels in accordance with the
present invention.
Scaffolding or biomaterial characteristics may enable cells to
attach and interact with the scaffolding or biomaterial material,
and/or may provide porous spaces into which cells can be entrapped.
In one embodiment, the porous scaffolds or biomaterials of the
present invention allow for the addition or deposition of one or
more populations or admixtures of cells on a biomaterial configured
as a porous scaffold (e.g., by attachment of the cells) and/or
within the pores of the scaffold (e.g., by entrapment of the
cells). In another embodiment, the scaffolds or biomaterials allow
or promote for cell:cell and/or cell:biomaterial interactions
within the scaffold to form constructs as described herein.
In one embodiment, the biomaterial used in accordance with the
present invention is comprised of hyaluronic acid (HA) in hydrogel
form, containing HA molecules ranging in size from 5.1 kDA to
>2.times.10.sup.6 kDa. In another embodiment, the biomaterial
used in accordance with the present invention is comprised of
hyaluronic acid in porous foam form, also containing HA molecules
ranging in size from 5.1 kDA to >2.times.10.sup.6 kDa. In yet
another embodiment, the biomaterial used in accordance with the
present invention is comprised of of a poly-lactic acid (PLA)-based
foam, having an open-cell structure and pore size of about 50
microns to about 300 microns. In yet another embodiment, the
specific cell populations, preferentially B2 but also B4, provide
directly and/or stimulate synthesis of high molecular weight
Hyaluronic Acid through Hyaluronic Acid Synthase-2 (HAS-2),
especially after intra-renal implantation.
Those of ordinary skill in the art will appreciate that other types
of synthetic or naturally-occurring materials known in the art may
be used to form scaffolds as described herein.
In one aspect, the present invention provides constructs as
described herein made from the above-referenced scaffolds or
biomaterials.
Constructs
In one aspect, the invention provides implantable constructs having
one or more of the cell populations described herein for the
treatment of kidney disease, anemia, or EPO deficiency in a subject
in need. In one embodiment, the construct is made up of a
biocompatible material or biomaterial, scaffold or matrix composed
of one or more synthetic or naturally-occurring biocompatible
materials and one or more cell populations or admixtures of cells
described herein deposited on or embedded in a surface of the
scaffold by attachment and/or entrapment. In certain embodiments,
the construct is made up of a biomaterial and one or more cell
populations or admixtures of cells described herein coated with,
deposited on, deposited in, attached to, entrapped in, embedded in,
or combined with the biomaterial component(s). Any of the cell
populations described herein, including enriched cell populations
or admixtures thereof, may be used in combination with a matrix to
form a construct.
In another embodiment, the deposited cell population or cellular
component of the construct is a first kidney cell population
enriched for oxygen-tunable EPO-producing cells. In another
embodiment, the first kidney cell population contains glomerular
and vascular cells in addition to the oxygen-tunable EPO-producing
cells, in one embodiment, the first kidney cell population is a B4'
cell population. In one other embodiment, the deposited cell
population or cellular component(s) of the construct includes both
the first enriched renal cell population and a second renal cell
population. In some embodiments, the second cell population is not
enriched for oxygen-tunable EPO producing cells. In another
embodiment, the second cell population is enriched for renal
tubular cells. In another embodiment, the second cell population is
enriched for renal tubular cells and contains collecting duct
epithelial cells. In other embodiments, the renal tubular cells are
characterized by the expression of one or more tubular cell markers
that may include, without limitation, megalin cubilin, hyaluronic
acid synthase 2 (HAS2), Vitamin D3 25-Hydroxylase (CYP2D25),
N-cadherin (Ncad), E-cadherin (Ecad), Aquaporin-1 (Aqp1),
Aquaporin-2 (Aqp2), RAB17, member RAS oncogene family (Rab17), GATA
binding protein 3 (Gata3). FXYD domain-containing ion transport
regulator 4 (Fxvd4), solute carrier family 9 (sodium/hydrogen
exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3 family,
member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3
(Aldh1a3), and Calpain-8 (Capn8).
In one embodiment, the cell populations deposited on or combined
with biomaterials or scaffolds to form constructs of the present
invention are derived from a variety of sources, such as autologous
sources. Non-autologous sources are also suitable for use,
including without limitation, allogeneic, or syngeneic (autogenic
or isogeneic) sources.
Those of ordinary skill in the art will appreciate there are
several suitable methods for depositing or otherwise combining cell
populations with biomaterials to form a construct.
In one aspect, the constructs of the present invention are suitable
for use in the methods of use described herein. In one embodiment,
the constructs are suitable for administration to a subject in need
of treatment for a kidney disease of any etiology, anemia, or EPO
deficiency of any etiology. In other embodiments, the constructs
are suitable for administration to a subject in need of an
improvement in or restoration of erythroid homeostasis. In another
embodiment, the constructs are suitable for administration to a
subject in need of improved kidney function.
In yet another aspect, the present invention provides a construct
for implantation into a subject in need of improved kidney function
comprising: a) a biomaterial comprising one or more biocompatible
synthetic polymers or naturally-occurring proteins or peptides;
and
b) an admixture of mammalian renal cells derived from a subject
having kidney disease comprising a first cell population, B2,
comprising an isolated, enriched population of tubular cells having
a density between 1.045 g/mL, and 1.052 g/mL and a second cell
population, B4', comprising erythropoietin (EPO)-producing cells
and vascular cells but depleted of glomerular cells having a
density between 1.063 g/mL, and 1.091 g/mL coated with, deposited
on or in, entrapped in, suspended in, embedded in and/or otherwise
combined with the biomaterial. In certain embodiments, the
admixture does not include a B1 cell population comprising large
granular cells of the collecting duct and tubular system having a
density of <1.045 g/ml, or a B5 cell population comprising
debris and small cells of low granularity and viability with a
density>1.091 g/ml.
In one embodiment, the construct includes a B4' cell population
which is characterized by expression of a vascular marker. In some
emodiments, the B4' cell population is not characterized by
expression of a glomerular marker. In certain embodiments, the
admixture is capable of oxygen-tunable erythropoietin (EPO)
expression. In all embodiments, the admixture may be derived from
mammalian kidney tissue or cultured kidney cells.
In one embodiment, the construct includes a biomaterial configured
as a three-dimensional (3-D) porous biomaterial suitable for
entrapment and/or attachment of the admixture. In another
embodiment, the construct includes a biomaterial configured as a
liquid or semi-liquid gel suitable for embedding, attaching,
suspending, or coating mammalian cells. In yet another embodiment,
the construct includes a biomaterial configured comprised of a
predominantly high-molecular weight species of hyaluronic acid (HA)
in hydrogel form. In another embodiment, the construct includes a
biomaterial comprised of a predominantly high-molecular weight
species of hyaluronic acid in porous foam form. In yet another
embodiment, the construct includes a biomaterial comprised of a
poly-lactic acid-based foam having pores of between about 50
microns to about 300 microns. In still another embodiment, the
construct includes one or more cell populations that may be derived
from a kidney sample that is autologous to the subject in need of
improved kidney function. In certain embodiments, the sample is a
kidney biopsy. In some emodiments, the subject has a kidney
disease. In yet other embodiments, the cell population is derived
from a non-autologous kidney sample. In one embodiment, the
construct provides erythroid homeostasis.
Secreted Products
In one other aspect, the present invention concerns products
secreted from an enriched renal cell population or admixture of
enriched renal cell populations, as described herein. In one
embodiment, the products include one or more of the following:
paracrine factors, endocrine factors, juxtacrine factors, and
vesicles. The vesicles may include one or more of the following:
paracrine factors, endocrine factors, juxtacrine factors,
microvesicles, exosomes, and RNA. The secreted products may also
include products that are not within microvesicles including,
without limitation, paracrine factors, endocrine factors,
juxtacrine factors, and RNA. For example, extracellular miRNAs have
been detected externally to vesicles (Wang et al., Nuc Acids Res
2010, 1-12 doi:10.1093/nar/gkq601, Jul. 7, 2010). The secreted
products may also be referred to as cell-derived products, e.g.,
cell-derived vesicles.
In one other embodiment, the secreted products may be part of a
vesicle derived from renal cells. The vesicles may be capable of
delivering the factors to other destinations. In one embodiment,
the vesicles are secreted vesicles. Several types of secreted
vesicles are contemplated including, without limitation, exosomes,
microvesicles, ectosomes, membrane particles, exosome-like
vesicles, and apoptotic vesicles (Thery et al. 2010. Nat. Rev.
Immunol. 9:581-593). In one embodiment, the secreted vesicles are
exosomes. In one other embodiment, the secreted vesicles are
microvesicles. In one other embodiment, the secreted vesicles
contain or comprise one or more cellular components. The components
may be one or more of the following: membrane lipids, RNA,
proteins, metabolities, cytosolic components, and any combination
thereof. In a preferred embodiment, the secreted vesicles comprise,
consist of, or consist essentially of microRNAs. Preferably, the
miRNAs are human miRNAs. In one embodiment, one or more miRNAs are
selected from the group consisting of miR-30b-5p, miR-449a,
miR-146a, miR-130a, miR-23b, miR-21, miR-124, and miR-151. In one
other embodiment, one or more miRNAs may be selected from the group
consisting of let-7a-1; let-7a-2; let-7a-3; let-7b; let-7c; let-7d;
let-7e; let-7f-1; let-7f-2; let-7 g; let-7i; mir-1-1; mir-1-2;
mir-7-1; mir-7-2; mir-7-3; mir-9-1; mir-9-2; mir-9-3; mir-10a;
mir-10b; mir-15a; mir-15b; mir-16-1; mir-16-2; mir-17; mir-18a;
mir-18b; mir-19a; mir-19b-1; mir-20a; mir-20b; mir-21; mir-22;
mir-23a; mir-23b; mir-23c; mir-24-1; mir-24-2; mir-25; mir-26a-1;
mir-26a-2; mir-26b; mir-27a; mir-27b; mir-28; mir-29a; mir-29b-1;
mir-29b-2; mir-29c; mir-30a; mir-30b; mir-30c-1; mir-30c-2;
mir-30d; mir-30e; mir-31; mir-32; mir-33a; mir-33b; mir-34a;
mir-34b; mir-34c; mir-92a-1; mir-92a-2; mir-92b; mir-93; mir-95;
mir-96; mir-98; mir-99a mir-99b; mir-100; mir-101-1; mir-101-2;
mir-103-1; mir-103-1-as; mir-103-2; mir-103-2-as; mir-105-1;
mir-105-2; mir-106a; mir-106b; mir-107; mir-122; mir-124-1;
mir-124-2; mir-124-3; mir-125a; mir-125b-1; mir-125b-2; mir-126;
mir-127; mir-128-1; mir-128-2; mir-129-1; mir-129-2; mir-130a;
mir-130b; mir-132; mir-132; mir-133a-1; mir-133a-2; mir-133b;
mir-134; mir-135a-1; mir-135a-2; mir-135b; mir-136 MI101351120;
mir-137; mir-138-1; mir-138-2; mir-139; mir-140; mir-141; mir-142;
mir-143; mir-144; mir-145; mir-146a; mir-146b; mir-147; mir-147b;
mir-148a; mir-148b; mir-149; mir-150; mir-151; mir-152; mir-153-1;
mir-153-2; mir-154; mir-155; mir-181a-1; mir-181a-2; mir-181b-1;
mir-181 b-2; mir-181c; mir-181 d; mir-182; mir-183; mir-184;
mir-185; mir-186; mir-187; mir-188; mir-190; mir-190 mir-191;
mir-192; mir-193a; mir-193b; mir-194-1; mir-194-2; mir-195;
mir-196a-1; mir-196a-2; mir-196b; mir-197; mir-198; mir-199a-1;
mir-199a-2; mir-199b; mir-200a; mir-200b; mir-200c; mir-202;
mir-203; mir-204; mir-205; mir-206; mir-208a; mir-208b; mir-210;
mir-211; mir-212; mir-214; mir-215; mir-216a; mir-216b; mir-217;
mir-218-1; mir-218-2; mir-219-1; mir-219-2; mir-221; mir-222;
mir-223; mir-224; mir-296; mir-297; mir-298; mir-299; mir-300;
mir-301a; mir-301b; mir-302a; mir-302b; mir-302c; mir-302d;
mir-302e; mir-302f; mir-320a; mir-320b-1; mir-320b-2; mir-320c-1;
mir-320c-2; mir-320d-1; mir-320d-2; mir-320e; mir-323; mir-323b;
mir-324; mir-325; mir-326; mir-328; mir-329-1; mir-329-2; mir-330;
mir-331; mir-335; mir-337; mir-338; mir-339; mir-340; mir-341,
mir-345; mir-346; mir-361; mir-362; mir-363; mir-365-1; mir-365-2;
mir-367; mir-369; mir-370; mir-37; mir-372; mir-373; mir-374a;
mir-374b; mir-374c; mir-375; mir-376a-1; mir-376a-2; mir-376b;
mir-376c; mir-377; mir-378; mir-378b; mir-378c; mir-379; mir-380;
mir-381; mir-382; mir-383; mir-384; mir-409; mir-410; mir-411;
mir-412; mir-421; mir-422a; mir-423; mir-424; mir-425; mir-429;
mir-431; mir-432; mir-433; mir 448; mir-449a; mir-449b; mir-449c;
mir-450a-1; mir-450a-2; mir-450b; mir-451; mir-452; mir-454;
mir-455; mir-466; mir-483; mir-484; mir-485; mir-486; mir-487a;
mir-487b; mir-488; mir-489; mir-490; mir-491; mir-492; mir-493;
mir-494; mir-495; mir-496; mir-498; mir-499; mir-500a; mir-500b;
mir-501; mir-502; mir-503; mir-504; mir-505; mir-506; mir-507;
mir-508; mir-509-1; mir-509-2; mir-509-3; mir-510; mir-511-1;
mir-511-2; mir-512-1; mir-512-2; mir-513a-1; mir-513a-2; mir-513b;
mir-513c; mir-514-1; mir-514-2; mir-514-3; mir-514b; mir-515-1;
mir-515-2; mir-516a-1; mir-516a-2; mir-516b-1; mir-516b-2;
mir-517a; mir-517b; mir-517c; mir-518a-1; mir-518a-2; mir-518b;
mir-518c; mir-518d; mir-518e; mir-518f; mir-519a-1; mir-519a-2;
mir-519b; mir-519c; mir-519d; mir-519c; mir-520a; mir-520b;
mir-520c; mir-520d; mir-520e; mir-520f; mir-520 g; mir-520b;
mir-521-1; mir-521-2; mir-522; mir-523; mir-524; mir-525;
mir-526a-1; mir-526a-2; mir-526b; mir-527; mir-532; mir-539;
mir-541; mir-542; mir-543; mir-544; mir-544b; mir-545; mir-548a-1;
mir-548a-2; mir-548a-3; mir-548aa-1; mir-548aa-2; mir-548b:
mir-548c; mir-548d-1: mir-548d-2; mir-548e; mir-548f-1; mir-548f-2;
mir-548f-3; mir-548f-4; mir-548f-5; mir-548 g; mir-548h-1;
mir-548h-2; mir-548h-3; mir-548h-4; mir-548i-1; mir-548i-2;
mir-548i-3; mir-548j: mir-548k; mir-548l; mir-548m; mir-548n;
mir-548o; mir-548p; mir-548s; mir-548t; mir-548u; mir-548v;
mir-548w; mir-548x; mir-548y; mir-548z; mir-549; mir-550a-1;
mir-550a-2; mir-550b-1; mir-550-2; mir-551a; mir-551b; mir-552;
mir-553; mir-554; mir-555; mir-556; mir-557; mir-558; mir-559;
mir-561; mir-562; mir-563; mir-564; mir-566; mir-567; mir-568;
mir-569; mir-570; mir-571; mir-572; mir-573; mir-574; mir-575;
mir-576; mir-577; mir-578; mir-579; mir-580; mir-581; mir-582;
mir-583; mir-584; mir-585; mir-586; mir-587; mir-588; mir-589;
mir-590; mir-591; mir-592; mir-593; mir-595; mir-596; mir-597;
mir-598; mir-599; mir-600; mir-601; mir-602; mir-603; mir-604;
mir-605; mir-606; mir-607; mir-608; mir-609; mir-610; mir-611;
mir-612; mir-613; mir-614; mir-615; mir-616; mir-617; mir-618;
mir-619; mir-620; mir-621; mir-622; mir-623; mir-624; mir-625;
mir-626; mir-627; mir-628; mir-629; mir-630, mir-631; mir-632;
mir-633; mir-634; mir-635; mir-636; mir-637; mir-638; mir-639;
mir-640; mir-641; mir-642a; mir-642b; mir-643; mir-644; mir-645;
mir-646; mir-647; mir-648; mir-649; mir-650; mir-651; mir-652;
mir-653; mir-654; mir-655; mir-656; mir-657; mir-658; mir-659;
mir-660; mir-661; mir-662; mir-663; mir-663b; mir-664; mir-665;
mir-668, mir-670; mir-671; mir-675; mir-676; mir-711; mir-718;
mir-720; mir-744; mir-758; mir-759; mir-760; mir-761; mir-762;
mir-764; mir-765; mir-766; mir-767; mir-769; mir-770; mir-802;
mir-873 mir-874; mir-875; mir-876; mir-877; mir-885; mir-887;
mir-888; mir-889; mir-890; mir-891a; mir-891b; mir-892a; mir-892b;
mir-920; mir-921; mir-922; mir-924; mir-933; mir-934; mir-935;
mir-936; mir-937; mir-938; mir-939; mir-940; mir-941-1; mir-941-2;
mir-941-3; mir-941-4; mir-942; mir-942; mir-943 mir-944; mir-1178;
mir-1179; mir-1180; mir-1181; mir-1182; mir-1183; mir-1184-1;
mir-1184-2; mir-1184-3; mir-1185-1; mir-1185-2; mir-1193; mir-1197;
mir-1200; mir-1202; mir-1203; mir-1204; mir-1205; mir-1206;
mir-1207; mir-1208; mir-1224; mir-1225; mir-1226; mir-1227;
mir-1228; mir-1229; mir-1231; mir-1233-1; mir-1233-2; mir-1234;
mir-1236; mir-1237; mir-1238; mir-1243; mir-1244-1; mir-1244-2;
mir-1244-3; mir-1245; mir-1246; mir-1247; mir-1248; mir-1249;
mir-1250; mir-1251; mir-1252; mir-1253; mir-1254; mir-1255a;
mir-1255b-1; mir-1255b-2; mir-1256; mir-1257; mir-1258; mir-1260;
mir-1260b; mir-1261; mir-1262; mir-1263; mir-1264; mir-1265;
mir-12661, mir-1267; mir-1268; mir-1269; mir-1270-1; mir-1270-2;
mir-1271; mir-1272; mir-1273; mir-1273c; mir-1273d; mir-1273e;
mir-1274a; mir-1274b; mir-1275; mir-1276; mir-1277; mir-1278;
mir-1279; mir-1280; mir-1281; mir-1282; mir-1283-1; mir-1283-2;
mir-1284; mir-1285-1; mir-1285-2; mir-1286; mir-1287; mir-1288;
mir-1289-1; mir-1289-2; mir-1290; mir-1291; mir-1292; mir-1293;
mir-1294; mir-1295; mir-1296; mir-1297; mir-1298; mir-1299;
mir-1301; mir-1302-1; mir-1302-10; mir-1302-11; mir-1302-2;
mir-1302-3; mir 1302-4; mir-1302-5; mir-1302-6; mir-1302-7;
mir-1302-8; mir-1302-9; mir-1303; mir-1304; mir-1305; mir-1306;
mir-1307; mir-1321; mir-1322; mir-1323; mir-1324; mir-1468;
mir-1469; mir-1470; mir-1471; mir-1537; mir-1538; mir-1539;
mir-1825; mir-1827; mir-1908; mir-1909; mir-1910; mir-1911;
mir-1912; mir-1913; mir-1914; mir-1915; mir-1972-1; mir-1972-2;
mir-1973; mir-1976; mir-2052; mir-2053; mir-2054; mir-2110;
mir-2113; mir-2114; mir-2115; mir-2116; mir-2117; mir-2276;
mir-2277; mir-2278; mir-2355; mir-2861; mir-2909; mir-3065;
mir-3074; mir-3115; mir-3116-1; mir-3116-2; mir-3117; mir-3118-1;
mir-3118-2; mir-3118-3; mir-3118-4; mir-3118-5; mir-3118-6;
mir-3119-1; mir-3119-2; mir-3120; mir-3121; mir-3122; mir-3123;
mir-3124; mir-3125; mir-3126; mir-3127; mir-3128; mir-3129;
mir-3130-1; mir-3130-2; mir-3131; mir-3132; mir-3133; mir-3134;
mir-3135; mir-3136; mir-3137; mir-3138; mir-3139; mir-3140;
mir-3141; mir-3142; mir-3143; mir-3144; mir-3145; mir-3146;
mir-3147; mir-3148; mir-3149; mir-3150; mir-3151; mir-3152;
mir-3153; mir-3154; mir-3155; mir-3156-1; mir-3156-2; mir-3156-3;
mir-3157; mir-3158-1; mir-3158-2; mir-3159; mir-3160-1; mir-3160-2;
mir-3161; mir-3162; mir-3163; mir-3164; mir-3165; mir-3166;
mir-3167; mir-3168; mir-3169; mir-3170; mir-3171; mir-3173;
mir-3174; mir-3175; mir-3176; mir-3177; mir-3178; mir-3179-1;
mir-3179-2; mir-3179-3; mir-3180-1; mir-3180-2; mir-3180-3;
mir-3180-4; mir-3180-5; mir-3181; mir-3182; mir-3183; mir-3184;
mir-3185; mir-3186; mir-3187; mir-3188; mir-3189; mir-3190;
mir-3191; mir-3192; mir-3193; mir-3194; mir-3195; mir-3196;
mir-3197; mir-3198; mir-3199-1; mir-3199-2; mir-3200; mir-3201;
mir-3202-1; mir-3202-2; mir-3605; mir-3606; mir-3607; mir-3609;
mir-3610; mir-3611; mir-3612; mir-3613; mir-3614; mir-3615;
mir-3616; mir-3617; mir-3618; mir-3619; mir-3620; mir-3621;
mir-3622a; mir-3622b; mir-3646; mir-3647; mir-3648; mir-3649;
mir-3650; mir-3651; mir-3652; mir-3653; mir-3654; mir-3655;
mir-3656mir-3657; mir-3658; mir-3659; mir-3660; mir-3661; mir-3662;
mir-3663; mir-3664; mir-3665; mir-3666; mir-3667; mir-3668;
mir-3669; mir-3670; mir-3670; mir-3671; mir-3671; mir-3673;
mir-3673; mir-3675; mir-3675; mir-3676; mir-3663; mir-3677;
mir-3678; mir-3679; mir-3680; mir-3681; mir-3682; mir-3683;
mir-3684; mir-3685; mir-3686; mir-3687; mir-3688; mir-3689a;
mir-3689b; mir-3690; mir-3691; mir-3692; mir-3713; mir-3714;
mir-3907; mir-3908; mir-3909; mir-3910-1; mir-3910-2; mir-3911;
mir-3912; mir-3913-1; mir-3913-2; mir-3914-1; mir-3914-2; mir-3915;
mir-3916; mir-3917; mir-3918; mir-3919; mir-3920; mir-3921;
mir-3922; mir-3923; mir-3924; mir-3925; mir-3926-1; mir-3926-2;
mir-3927; mir-3928; mir-3929; mir-3934; mir-3935; mir-3936;
mir-3937; mir-3938; mir-3939; mir-3940; mir-3941; mir-3942;
mir-3943; mir-3944; mir-3945; mir-4251; mir-4252; mir-4253;
mir-4254; mir-4255; mir-4256; mir-4257; mir-4258; mir-4259;
mir-4260; mir-4261; mir-4262; mir-4263; mir-4264; mir-4265;
mir-4266; mir-4267; mir-4268; mir-4269; mir-4270; mir-4271;
mir-4272; mir-4273; mir-4274; mir-4275; mir-4276; mir-4277;
mir-4278; mir-4279; mir-4280; mir-4281; mir-4282; mir-4283-1;
mir-4283-2; mir-4284; mir-4285; mir-4286; mir-4287; mir-4288;
mir-4289; mir-4290; mir-4291; mir-4292; mir-4293; mir-4294;
mir-4295; mir-4296; mir-4297; mir-4298; mir-4299; mir-4300;
mir-4301; mir-4302; mir-4303; mir-4304; mir-4305; mir-4306;
mir-4307; mir-4308; mir-4309; mir-4310; mir-4311; mir-4312;
mir-4313; mir-4314; mir-4315-1; mir-4315-2; mir-4316; mir-4317;
mir-4318; mir-4319; mir-4320; mir-4321; mir-4322; mir-4323;
mir-4324; mir-4325; mir-4326; mir-4327; mir-4328; mir-4329;
mir-4329; and mir-4330.
The present invention relates to cell-derived or secreted miRNAs
obtainable from the cell populations or constructs described
herein. In one embodiment, one or more of the individual miRNAs may
be used to provide a regenerative effect to a native kidney.
Combinations of the individual miRNAs may be suitable for providing
such an effect. Exemplary combinations include two or more of the
following: miR-21; miR-23a; miR-30c; miR-1224; miR-23b; miR-92a;
miR-100; miR-125b-5p; miR-195; miR-10a-5p; and any combination
thereof. Another exemplary combination includes two or more of the
following: miR-30b-5p, miR-449a, miR-146a, miR-130a, miR-23b,
miR-21, miR-124, miR-151, and any combination thereof. In one
embodiment, the combination of miRNAs may include 2, 3, 4, 5, 6, 7,
8, 9, 10, or more individual miRNAs. Those of ordinary skill in the
are will appreciate that other miRNAs and combinations of mirRNAs
may be suitable for use in the present invention. Sources of
additional miRNAs include miRBase at http://mirbase.org, which is
hosted and maintained in the Faculty of Life Sciences at the
University of Manchester.
In one embodiment, the secreted products comprise paracrine
factors. In general, paracrine factors are molecules synthesized by
a cell that call diffuse over small distances to induce or effect
changes in a neighboring cell, i.e., a paracrine interaction. The
diffusable molecules are referred to as paracrine factors.
In yet another embodiment, the present invention concerns a
composition of one or more isolated renal-cell derived secreted
vesicles, as described herein. Those of ordinary skill in the art
will appreciate that various types of compositions containing the
secreted vesicles will be suitable.
In another aspect, the present invention provides methods of
preparing renal cell secreted products, e.g., vesicles. In one
embodiment, the method includes the steps of providing a renal cell
population, including admixtures of one or more enriched renal cell
populations. In another embodiment, the method further includes the
step of culturing the population under suitable conditions. The
conditions may be low oxygen conditions. In another embodiment, the
method further includes the step of isolating the secreted products
from the renal cell population. The secreted vesicles may be
obtained from the cell culture media of the cell population. In one
other embodiment, the renal cells are characterized by vesicle
production and/or secretion that is bioresponsive to oxygen levels,
such that a reduction in the oxygen tension of the culture system
results in an induction of vesicle production and/or secretion. In
one embodiment, the vesicle production and/or secretion is induced
when the cell population is cultured under conditions where the
cells are subjected to a reduction in available oxygen levels in
the culture system as compared to a cell population cultured at
normal atmospheric (.about.21%) levels of available oxygen. In one
embodiment, the cell populations cultured in lower oxygen
conditions produce and/or secrete greater levels of vesicles
relative to cell populations cultured at normal oxygen conditions.
In general, the culturing of cells at reduced levels of available
oxygen (also referred to as hypoxic culture conditions) means that
the level of reduced oxygen is reduced relative to the culturing of
cells at normal atmospheric levels of available oxygen (also
referred to as normal or normoxic culture conditions). In one
embodiment, hypoxic cell culture conditions include culturing cells
at about less than 1% oxygen, about less than 2% oxygen, about less
than 3% oxygen, about less than 4% oxygen, or about less than 5%
oxygen. In another embodiment, normal or normoxic culture
conditions include culturing cells at about 10% oxygen, about 12%
oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about
16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen,
about 20% oxygen, or about 21% oxygen. In a preferred embodiment,
the method provides for the isolation of exosomes and/or
microvesicles from renal cells.
In one embodiment, the products are secreted from renal cells. The
products may be secreted from renal cells that are not on a
scaffold, e.g., the cells are not part of a construct as described
herein.
In another embodiment, the products are secreted by renal cells
that have been seeded on a scaffold, e.g., a construct. The
construct includes one or more enriched renal cell populations or
an admixture thereof that are directly seeded on or in a
scaffold.
In another aspect, the present invention provides in vitro methods
for screening/optimizing/monitoring the biotherapeutic efficacy of
one or more enriched renal cell populations, and admixtures or
constructs containing the same. In one embodiment, the method
includes the step of providing one or more test populations, test
admixture or test construct (the "test article"). In another
embodiment, the method includes the step of culturing the test
article under suitable conditions, as described herein. In one
other embodiment, the method includes the step of collecting cell
culture media from the cultured test article. This media may be
referred to as "conditioned media" and it is expected to contain
products secreted by the renal cells of the test article.
In one other aspect, the conditioned media may be used to conduct
one or more in vitro assays in order to test the biotherapeutic
efficacy of the test article. In one embodiment, the conditioned
media is subjected to an epithelial-mesenchymal transition (EMT)
assay. The assay may test for EMT induced by TGF.beta.1. Example 15
provides an exemplary protocol for this assay.
In another embodiment, the conditioned media is subjected to the
detection of RNAs, e.g., via PCR-based assays, and/or vesicles or
exosomes, e.g., via FACS. In one other embodiment, the conditioned
media is subjected to a signaling pathway assay, e.g., immune
response (e.g., NF.kappa.B), fibrotic response (PAI-1), and
angiogenesis. Examples 12-14 provides exemplary protocols for these
assays.
Methods of Use
In one aspect, the present invention provides methods for the
treatment of a kidney disease, anemia, or EPO deficiency in a
subject in need with the kidney cell populations and admixtures of
kidney cells described herein. In one embodiment, the method
comprises administering to the subject a composition that includes
a first kidney cell population enriched for EPO-producing cells. In
another embodiment, the first cell population is enriched for
EPO-producing cells, glomerular cells, and vascular cells. In one
embodiment, the first kidney cell population is a B4' cell
population. In another embodiment, the composition may further
include one or more additional kidney cell populations. In one
embodiment, the additional cell population is a second cell
population not enriched for EPO-producing cells. In another
embodiment, the additional cell population is a second cell
population not enriched for EPO-producing cells, glomerular cells,
or vascular cells. In another embodiment, the composition also
includes a kidney cell population or admixture of kidney cells
deposited in, deposited on, embedded in, coated with, or entrapped
in a biomaterial to form an implantable construct, as described
herein, for the treatment of a disease or disorder described
herein. In one embodiment, the cell populations are used alone or
in combination with other cells or biomaterials, e.g., hydrogels,
porous scaffolds, or native or synthetic peptides or proteins, to
stimulate regeneration in acute or chronic disease states.
In another aspect, the effective treatment of a kidney disease,
anemia, or EPO deficiency in a subject by the methods of the
present invention can be observed through various indicators of
erythropoiesis and/or kidney function. In one embodiment, the
indicators of erythroid homeostasis include, without limitation,
hematocrit (HCT), hemoglobin (HB), mean corpuscular hemoglobin
(MCH), red blood cell count (RBC), reticulocyte number,
reticulocyte %, mean corpuscular volume (MCV), and red blood cell
distribution width (RDW). In one other embodiment, the indicators
of kidney function include, without limitation, serum albumin,
albumin to globulin ratio (A/G ratio), serum phosphorous, serum
sodium, kidney size (measurable by ultrasound), serum calcium,
phosphorous:calcium ratio, serum potassium, proteinuria, urine
creatinine, serum creatinine, blood nitrogen urea (BUN),
cholesterol levels, triglyceride levels and glomerular filtration
rate (GFR). Furthermore, several indicators of general health and
well-being include, without limitation, weight gain or loss,
survival, blood pressure (mean systemic blood pressure, diastolic
blood pressure, or systolic blood pressure), and physical endurance
performance.
In another embodiment, an effective treatment is evidenced by
stabilization of one or more indicators of kidney function. The
stabilization of kidney function is demonstrated by the observation
of a change in an indicator in a subject treated by a method of the
present invention as compared to the same indicator in a subject
that has not been treated by a method of the present invention.
Alternatively, the stabilization of kidney function may be
demonstrated by the observation of a change in an indicator in a
subject treated by a method of the present invention as compared to
the same indicator in the same subject prior to treatment. The
change in the first indicator may be an increase or a decrease in
value. In one embodiment, the treatment provided by the present
invention may include stabilization of blood urea nitrogen (BUN)
levels in a subject where the BUN levels observed in the subject
are lower as compared to a subject with a similar disease state who
has not been treated by the methods of the present invention. In
one other embodiment, the treatment may include stabilization of
serum creatinine levels in a subject where the serum creatinine
levels observed in the subject are lower as compared to a subject
with a similar disease state who has not been treated by the
methods of the present invention. In another embodiment, the
treatment may include stabilization of hematocrit (HCT) levels in a
subject where the HCT levels observed in the subject are higher as
compared to a subject with a similar disease state who has not been
treated by the methods of the present invention. In another
embodiment, the treatment may include stabilization of red blood
cell (RBC) levels in a subject where the RBC levels observed in the
subject are higher as compared to a subject with a similar disease
state who has not been treated by the methods of the present
invention. Those of ordinary skill in the art will appreciate that
one or more additional indicators described herein or known in the
art may be measured to determine the effective treatment of a
kidney disease in the subject.
In another aspect, the present invention concerns a method of
providing erythroid homeostasis in a subject in need. In one
embodiment, the method includes the step of (a) administering to
the subject a renal cell population, e.g., B2 or B4', or admixture
of renal cells, e.g., B2/B4' and/or B2/B3, as described herein; and
(b) determining, in a biological sample from the subject, that the
level of an erythropoiesis indicator is different relative to the
indicator level in a control, wherein the difference in indicator
level (i) indicates the subject is responsive to the administering
step (a), or (ii) is indicative of erythroid homeostasis in the
subject. In another embodiment, the method includes the step of (a)
administering to the subject a composition comprising a renal cell
population or admixture of renal cells as described herein; and (b)
determining, in a biological sample from the subject, that the
level of an erythropoiesis indicator is different relative to the
indicator level in a control, wherein the difference in indicator
level (i) indicates the subject is responsive to the administering
step (s), or (ii) is indicative of erythroid homeostasis in the
subject. In another embodiment, the method includes the step of (a)
providing a biomaterial or biocompatible polymeric scaffold; (b)
depositing a renal cell population or admixture of renal cells of
the present invention on or within the biomaterial or scaffold in a
manner described herein to form an implantable construct; (c)
implanting the construct into the subject; and (d) determining, in
a biological sample from the subject, that the level of an
erythropoiesis indicator is different relative to the indicator
level in a control, wherein the difference in indicator level (i)
indicates the subject is responsive to the administering step (a),
or (ii) is indicative of erythroid homeostasis in the subject.
In another aspect, the present invention concerns a method of
providing both stabilization of kidney function and restoration of
erythroid homeostasis to a subject in need, said subject having
both a deficit in kidney function and an anemia and/or
EPO-deficiency. In one embodiment, the method includes the step of
administering a renal cell population or admixture of renal cells
as described herein that contain at least one of the following cell
types: tubular-derived cells, glomerulus-derived cells,
interstitium-derived cells, collecting duct-derived cells, stromal
tissue-derived cells, or cells derived from the vasculature. In
another embodiment, the population or admixture contains both
EPO-producing cells and tubular epithelial cells, the tubular cells
having been identified by at least one of the following markers:
megalin, cubilin, hyaluronic acid synthase 2 (HAS2), Vitamin D3
25-Hydroxylase (CYP2D2S), N-cadherin (Ncad), E-cadherin (Ecad),
Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogene
family (Rab17), GATA binding protein 3 (Gata3), FXYD
domain-containing ion transport regulator 4 (Fxyd4), solute carrier
family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde
dehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase
1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8). In this
embodiment, treatment of the subject would be demonstrated by an
improvement in at least one indicator of kidney function
concomitant with improvement in at least one indicator of
erythropoiesis, compared to either an untreated subject or to the
subject's pre-treatment indicators.
In one aspect, the present invention provides methods of (i)
treating a kidney disease, anemia, or an EPO-deficiency; (ii)
stabilizing kidney function, (iii) restoring erythroid homeostasis,
or (iv) any combination of thereof by administering a renal cell
population enriched for EPO-producing cells or admixture of renal
cells containing a cell population enriched for EPO-producing cells
as described herein, wherein the beneficial effects of the
administration are greater than the effects of administering a cell
population not enriched for EPO-producing cells. In another
embodiment, the enriched cell population provides an improved level
of serum blood urea nitrogen (BUN). In another embodiment, the
enriched cell population provides an improved retention of protein
in the serum. In another embodiment, the enriched cell population
provides improved levels of serum cholesterol and/or triglycerides.
In another embodiment, the enriched cell population provides an
improved level of Vitamin D. In one embodiment, the enriched cell
population provides an improved phosphorus:calcium ratio as
compared to a non-enriched cell population. In another embodiment,
the enriched cell population provides an improved level of
hemoglobin as compared to a non-enriched cell population. In a
further embodiment, the enriched cell population provides an
improved level of serum creatinine as compared to a non-enriched
cell population. In yet another embodiment, the enriched cell
population provides an improved level of hematocrit as compared to
a non-enriched cell population. In a further embodiment, the
enriched cell population provides an improved level of red blood
cell number (RBC #) as compared to a non-enriched cell population.
In one embodiment, the improved level of hematocrit is restored to
95% normal healthy level. In a further embodiment, the enriched
cell population provides an improved reticulocyte number as
compared to a non-enriched cell population. In other embodiments,
the enriched cell population provides an improved reticulocyte
percentage as compared to a non-enriched cell population. In yet
other embodiments, the enriched cell population provides an
improved level of red blood cell volume distribution width (RDW) as
compared to a non-enriched cell population. In yet another
embodiment, the enriched cell population provides an improved level
of hemoglobin as compared to a non-enriched cell population. In yet
another embodiment, the enriched cell population provides an
erythroietic response in the bone marrow, such that the marrow
cellularity is near-normal and the myeloiderythroid ratio is near
normal.
In another aspect, the present invention provides methods of (i)
treating a kidney disease, anemia, or an EPO-deficiency; (ii)
stabilizing kidney function, (iii) restoring erythroid homeostasis,
or (iv) any combination of thereof by administering an enriched
cell population, wherein the beneficial effects of administering a
renal cell population or admixture of renal cell populations
described herein are characterized by improved erythroid
homeostasis when compared to the beneficial effects provided by the
administering of recombinant EPO (rEPO). In one embodiment, the
population or admixture, when administered to a subject in need
provides improved erythroid homeostasis (as determined by
hematocrit, hemoglobin, or RBC #) when compared to the
administration of recombinant EPO protein. In one embodiment, the
population or admixture, when administered provides an improved
level of hematocrit. RBC, or hemoglobin as compared to recombinant
EPO, being no greater than about 10% lower or higher than
hematocrit in a control. In a further embodiment, a single dose or
delivery of the population or admixture, when administered provides
improvement in erythroid homeostasis (as determined by increase in
hematocrit, hemoglobin, or RBC #) in the treated subject for a
period of time that significantly exceeds the period of time that a
single dose or delivery of the recombinant EPO protein provides
improvement in erythroid homeostasis. In another embodiment, the
population or admixture, when administered at a dose described
herein does not result in hematocrit, hemoglobin, or RBC # greater
than about 110% of normal levels in matched healthy controls. In a
further embodiment, the population or admixture, when administered
at a dose described herein provides superior erythroid homeostasis
(as determined by hematocrit, hemoglobin, or RBC #) compared to
recombinant EPO protein delivered at a dose described herein. In
another embodiment, the recombinant EPO is delivered at a dose of
about 100 IU/kg, about 200 IU/kg, about 300 IU/kg, about 400 IU/kg,
or about 500 IU/kg. Those of ordinary skill in the art will
appreciate that other dosages of recombinant EPO known in the art
may be suitable.
Another embodiment of the present invention is directed to the use
of at least one cell population, including enriched cell
populations and admixtures thereof, described herein, or an
implantable construct described herein, or secreted products as
described herein, for the preparation of a medicament useful in the
treatment of a kidney disease, anemia, or EPO deficiency in a
subject in need, the providing of erythroid homeostasis in a
subject in need, the improvement of kidney function in a subject in
need, or providing a regenerative effect to a native kidney.
Another embodiment of the present invention is directed to the use
of specific enriched cell population(s) (described herein) for the
treatement of a kidney disease of a specific etiology, based on
selection of specific cell subpopulation(s) based on specific
verified therapeutic attributes.
In yet another aspect, the present invention provides a method of
treating a kidney disease in a subject in need, comprising:
administering to the subject a composition comprising an admixture
of mammalian renal cells comprising a first cell population, B2,
comprising an isolated, enriched population of tubular cells having
a density between 1.045 g/mL and 1.052 g/mL, and a second cell
population, B4', comprising erythropoietin (EPO)-producing cells
and vascular cells but depleted of glomerular cells having a
density between 1.063 g/mL and 1.091 g/mL, wherein the admixture
does not include a B1 cell population comprising large granular
cells of the collecting duct and tubular system having a density of
<1.045 g/ml, or a B5 cell population comprising debris and small
cells of low granularity and viability with a density>1.091
g/ml. In certain embodiments, the method includes determining in a
test sample from the subject that the level of a kidney function
indicator is different relative to the indicator level in a
control, wherein the difference in indicator level is indicative of
a reduction in decline, a stabilization, or an improvement of one
or more kidney functions in the subject. In one embodiment, the B4'
cell population used in the method is characterized by expression
of a vascular marker. In certain embodiments, the B4' cell
population used in the method is not characterized by expression of
a glomerular marker. In one embodiment, the admixture of cells used
in the method is capable of oxygen-tunable erythropoietin (EPO)
expression. In certain embodiments, the kidney disease to be
treated by the methods of the invention is accompanied by an
erythropoietin (EPO) deficiency. In certain embodiments, the EPO
deficiency is anemia. In some embodiments, the EPO deficiency or
anemia occurs secondary to renal failure in the subject. In some
other embodiments, the EPO deficiency or anemia occurs secondary to
a disorder selected from the group consisting of chronic renal
failure, primary EPO deficiency, chemotherapy or anti-viral
therapy, non-myeloid cancer, HIV infection, liver disease, cardiac
failure, rheumatoid arthritis, or multi-organ system failure. In
certain embodiments, the composition used in the method further
comprises a biomaterial comprising one or more biocompatible
synthetic polymers and/or naturally-occurring proteins or peptides,
wherein the admixture is coated with, deposited on or in, entrapped
in, suspended in, embedded in and/or otherwise combined with the
biomaterial. In certain embodiments, the admixture used in the
methods of the invention is derived from mammalian kidney tissue or
cultured mammalian kidney cells. In other embodiments, the
admixture is derived from a kidney sample that is autologous to the
subject in need. In one embodiment, the sample is a kidney biopsy.
In other embodiments, the admixture used in the methods of the
invention is derived from a non-autologous kidney sample.
In yet another aspect, the invention provides a use of the cell
preparations and admixtures thereof or an implantable construct of
the instant invention for the preparation of a medicament useful in
the treatment of a kidney disease, anemia or EPO deficiency in a
subject in need thereof.
In another aspect, the present invention provides methods for the
regeneration of a native kidney in a subject in need thereof. In
one embodiment, the method includes the step of administering or
implanting a cell population, admixture, or construct described
herein to the subject. A regenerated native kidney may be
characterized by a number of indicators including, without
limitation, development of function or capacity in the native
kidney, improvement of function or capacity in the native kidney,
and the expression of certain markers in the native kidney. In one
embodiment, the developed or improved function or capacity may be
observed based on the various indicators of erythroid homeostasis
and kidney function described above. In another embodiment, the
regenerated kidney is characterized by differential expression of
one or more stem cell markers. The stem cell marker may be one or
more of the following: SRY (sex determining region Y)-box 2 (Sox2);
Undifferentiated Embryonic Cell Transcription Factor (UTF1); Nodal
Homolog from Mouse (NODAL); Prominin 1 (PROM1) or CD133 (CD133);
CD24; and any combination thereof. In another embodiment, the
expression of the stem cell marker(s) is upregulated compared to a
control.
The cell populations described herein, including enriched cell
populations and admixtures thereof as well as constructs containing
the same, may be used to provide a regenerative effect to a native
kidney. The effect may be provided by the cells themselves and/or
by products secreted from the cells. The regenerative effect may be
characterized by one or more of the following: a reduction in
epithelial-mesenchymal transition (which may be via attenuation of
TGF-.beta. signalling); a reduction in renal fibrosis; a reduction
in renal inflammation; differential expression of a stem cell
marker in the native kidney; migration of implanted cells and/or
native cells to a site of renal injury, e.g., tubular injury,
engraftment of implanted cells at a site of renal injury, e.g.,
tubular injury; stabilization of one or more indicators of kidney
function (as described herein); restoration of erythroid
homeostasis (as described herein); and any combination thereof.
Methods of Monitoring Regeneration
In another aspect, the present invention provides a prognostic
method for monitoring regeneration of a native kidney following
administration or implantation of a cell population, admixture, or
construct described herein to the subject. In one embodiment, the
method includes the step of detecting the level of marker
expression in a test sample obtained from the subject and in a
control sample, wherein a higher level of expression of the marker
in the test sample, as compared to the control sample, is
prognostic for regeneration of the native kidney in the subject. In
another embodiment, the method includes the detection of expression
of one or more stem cell markers in the sample. The stem cell
marker may be selected from Sox2; UTF1; NODAL; CD133; CD24; and any
combination thereof. The detecting step may include determining
that expression of the stem cell marker(s) is upregulated or higher
in the test sample relative to a control sample, wherein the higher
level of expression is prognostic for regeneration of the subject's
native kidney. In one other embodiment, mRNA expression of the stem
cell marker(s) is detected. In other embodiments, the detection of
mRNA expression may be via a PCR-based method, e.g., qRT-PCR. In
situ hybridization may also be used for the detection of nRNA
expression.
In one other embodiment, polypeptide expression of the stem cell
marker(s) is detected. In another embodiment, polypeptide
expression is detected using an anti-stem cell marker agent. In one
other embodiment, the agent is an antibody against the marker. In
another embodiment, stem cell marker polypeptide expression is
detected using immunohistochemistry or a Western Blot.
Those of ordinary skill in the art will appreciate other methods
for detecting mRNA and/or polypeptide expression of markers.
In one embodiment, the detecting step is preceded by the step of
obtaining the test sample from the subject. In another embodiment,
the test sample is kidney tissue.
In one other aspect, the present invention provides the use of
markers, such as stem cell markers, as a surrogate marker for
regeneration of the native kidney. Such a marker could be used
independent of or in conjunction with an assessment of regeneration
based on whether function or capacity has been developed or
improved (e.g., indicators of erythroid homeostasis and kidney
function). Monitoring a surrogate marker over the time course of
regeneration may also serve as a prognostic indicator of
regeneration.
In another aspect, the invention provides methods for prognostic
evaluation of a patient following implantation or administration of
a cell population, admixture, or construct described herein. In one
embodiment, the method includes the step of detecting the level of
marker expression in a test sample obtained from said subject; (b)
determining the expression level in the test sample relative to the
level of marker expression relative to a control sample (or a
control reference value); and (c) predicting regenerative prognosis
of the patient based on the determination of marker expression
levels, wherein a higher level of expression of marker in the test
sample, as compared to the control sample (or a control reference
value), is prognostic for regeneration in the subject.
In another aspect, the invention provides methods for prognostic
evaluation of a patient following implantation or administration of
a cell population, admixture, or construct described herein. In one
embodiment, the method includes the steps of (a) obtaining a
patient biological sample; and (b) detecting stein cell marker
expression in the biological sample, wherein stein cell marker
expression is prognostic for regeneration of the native kidney in
the patient. In some embodiments, increased stem cell marker
expression in the patient biological sample relative to a control
sample (or a control reference value) is prognostic for
regeneration in the subject. In some embodiments, decreased stem
cell marker expression in the patient sample relative to the
control sample (or control reference value) is not prognostic for
regeneration in the subject. The patient sample may be a test
sample comprising a biopsy. The patient sample may be a bodily
fluid, such as blood or urine.
In one other aspect, the present invention provides prognostic
methods for monitoring regeneration of a native kidney following
administration or implantation of a cell population, admixture, or
construct described herein to the subject, in which a non-invasive
method is used. As an alternative to a tissue biopsy, a
regenerative outcome in the subject receiving treatment can be
assessed from examination of a bodily fluid, e.g., urine. It has
been discovered that microvesicles obtained from subject-derived
urine sources contain certain components including, without
limitation, specific proteins and miRNAs that are ultimately
derived from the renal cell populations impacted by treatment with
the cell populations of the present invention. These components may
include factors involved in stem cell replication and
differentiation, apoptosis, inflammation and immuno-modulation. A
temporal analysis of microvesicle-associated miRNA/protein
expression patterns allows for continuous monitoring of
regenerative outcomes within the kidney of subjects receiving the
cell populations, admixtures, or constructs of the present
invention. Example 17 describes exemplary protocols for analysis of
the urine of subjects.
These kidney-derived vesicles and/or the luminal contents of kidney
derived vesicles shed into the urine of a subject may be analyzed
for biomarkers indicative of regenerative outcome.
In one embodiment, the present invention provides methods of
assessing whether a kidney disease (KD) patient is responsive to
treatment with a therapeutic. The method may include the step of
determining or detecting the amount of vesicles or their luminal
contents in a test sample obtained from a KD patient treated with
the therapeutic, as compared to or relative to the amount of
vesicles in a control sample, wherein a higher or lower amount of
vesicles or their luminal contents in the test sample as compared
to the amount of vesicles or their luminal contents in the control
sample is indicative of the treated patient's responsiveness to
treatment with the therapeutic.
The present invention also provides a method of monitoring the
efficacy of treatment with a therapeutic in a KD patient. In one
embodiment, the method includes the step of determining or
detecting the amount of vesicles in a test sample obtained from a
KD patient treated with the therapeutic, as compared to or relative
to the amount of vesicles or their luminal contents in a control
sample, wherein a higher or lower amount of vesicles or their
luminal contents in the test sample as compared to the amount of
vesicles or their luminal contents in the control sample is
indicative of the efficacy of treatment with the therapeutic in the
KD patient.
The present invention also provides a method of identifying an
agent as a therapeutic effective to treat kidney disease (KD) in a
patient subpopulation. In one embodiment, the method includes the
step of determining a correlation between efficacy of the agent and
the presence of an amount of vesicles in samples from the patient
subpopulation as compared to the amount of vesicles or their
luminal contents in a sample obtained from a control sample,
wherein a higher or lower amount of vesicles or their luminal
contents in the samples from the patient subpopulation as compared
to the amount of vesicles or their luminal contents in the control
sample is indicative that the agent is effective to treat KD in the
patient subpopulation.
The present invention provides a method of identifying a patient
subpopulation for which an agent is effective to treat kidney
disease (KD). In one embodiment, the method includes the step of
determining a correlation between efficacy of the agent and the
presence of an amount of vesicles or their luminal contents in
samples from the patient subpopulation as compared to the amount of
vesicles or their luminal contents in a sample obtained from a
control sample, wherein a higher or lower amount of vesicles in the
samples from the patient subpopulation as compared to the amount of
vesicles or their luminal contents in the control sample is
indicative that the agent is effective to treat KD in the patient
subpopulation.
The determining or detecting step may include analyzing the amount
of miRNA or other secreted products that may exist in the test
sample (see Example 17).
The non-invasive prognostic methods may include the step of
obtaining a urine sample front the subject before and/or after
administration or implantation of a cell population, admixture, or
construct described herein. Vesicles and other secreted products
may be isolated from the urine samples using standard techniques
including without limitation, centriguation to remove unwanted
debris (Zhou et al. 2008. Kidney Int. 74(5):613-621; Skog et al.
U.S. Published Patent Application No. 20110053157, each of which is
incorporated herein by reference in its entirety).
The present invention relates to non-invasive methods to detect
regenerative outcome in a subject following treatment. The methods
involve detection of vesicles or their luminal contents in urine
from a treated subject. The luminal contents may be one or more
miRNAs. The detection of combinations or panels of the individual
miRNAs may be suitable for such prognostic methods. Exemplary
combinations include two or more of the following: miR-24; miR-195;
miR-871; miR-30b-5p; miR-19b; miR-99a; miR-429, let-7f; miR-200a;
miR-324-5p; miR-10a-5p; and any combination thereof. In one
embodiment, the combination of miRNAs may include 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, or more individual miRNAs. Those of ordinary skill in
the art will appreciate that other miRNAs and combinations of
miRNAs may be suitable for use in such prognostic methods. Sources
of additional miRNAs include miRBase at http://mirbase.org, which
is hosted and maintained in the Faculty of Life Sciences at the
University of Manchester.
Those of skill in the art will appreciate that the prognostic
methods for detecting regeneration may be suitable for subjects
treated with other therapeutics known in the art, apart from the
cell populations and constructs described herein.
In some embodiments, the determining step comprises the use of a
software program executed by a suitable processor for the purpose
of (i) measuring the differential level of marker expression (or
vesicles/vesicle contents) in a test sample and a control; and/or
(ii) analyzing the data obtained from measuring differential level
of marker expression in a test sample and a control. Suitable
software and processors are well known in the art and are
commercially available. The program may be embodied in software
stored on a tangible medium such as CD-ROM, a floppy disk, a hard
drive, a DVD, or a memory associated with the processor, but
persons of ordinary skill in the art will readily appreciate that
the entire program or parts thereof could alternatively be executed
by a device other than a processor, and/or embodied in firmware
and/or dedicated hardware in a well known manner.
Following the determining step, the measurement results, findings,
diagnoses, predictions and/or treatment recommendations are
typically recorded and communicated to technicians, physicians
and/or patients, for example. In certain embodiments, computers
will be used to communicate such information to interested parties,
such as, patients and/or the attending physicians. In some
embodiments, the assays will be performed or the assay results
analyzed in a country or jurisdiction which differs from the
country or jurisdiction to which the results or diagnoses are
communicated.
In a preferred embodiment, a prognosis, prediction and/or treatment
recommendation based on the level of marker expression measured in
a test subject having a differential level of marker expression is
communicated to the subject as soon as possible after the assay is
completed and the prognosis and/or prediction is generated. The
results and/or related information may be communicated to the
subject by the subject's treating physician. Alternatively, the
results may be communicated directly to a test subject by any means
of communication, including writing, electronic forms of
communication, such as email, or telephone. Communication may be
facilitated by use of a computer, such as in case of email
communications. In certain embodiments, the communication
containing results of a prognostic test and/or conclusions drawn
from and/or treatment recommendations based on the test, may be
generated and delivered automatically to the subject using a
combination of computer hardware and software which will be
familiar to artisans skilled in telecommunications. One example of
a healthcare-oriented communications system is described in U.S.
Pat. No. 6,283,761; however, the present invention is not limited
to methods which utilize this particular communications system. In
certain embodiments of the methods of the invention, all or some of
the method steps, including the assaying of samples, prognosis
and/or prediction of regeneration, and communicating of assay
results or prognoses, may be carried out in diverse (e.g., foreign)
jurisdictions.
In another aspect, the prognostic methods described herein provide
information to an interested party concerning the regenerative
success of the implantation or administration.
In all embodiments, the methods of providing a regenerated kidney
to a subject in need of such treatment as described herein may
include the post-implantation step of prognostic evaluation of
regeneration as described above.
Methods and Routes of Administration
The cell preparations and/or constructs of the instant invention
can be administered alone or in combination with other bioactive
components.
The therapeutically effective amount of the renal cell populations
or admixtures of renal cell populations described herein can range
from the maximum number of cells that is safely received by the
subject to the minimum number of cells necessary for treatment of
kidney disease, stabilization, reduced rate-of-decline, or
improvement of one or more kidney functions. In certain
embodiments, the methods of the present invention provide the
administration of renal cell populations or admixtures of renal
cell populations described herein at a dosage of about 10,000
cells/kg, about 20,000 cells/kg, about 30,000 cells/kg, about
40,000 cells/kg, about 50,000 cells/kg, about 100,000 cells/kg,
about 200,000 cells/kg, about 300,000 cells/kg, about 400,000
cells/kg, about 500,000 cells/kg, about 600,000 cells/kg, about
700,000 cells/kg, about 800,000 cells/kg, about 900,000 cells/kg,
about 1.1.times.10.sup.6 cells/kg, about 1.2.times.10.sup.6
cells/kg, about 1.3.times.10.sup.6 cells/kg, about
1.4.times.10.sup.6 cells/kg, about 1.5.times.10.sup.6 cells/kg,
about 1.6.times.10.sup.6 cells/kg, about 1.7.times.10.sup.6
cells/kg, about 1.8.times.10.sup.6 cells/kg, about
1.9.times.10.sup.6 cells/kg, about 2.1.times.10.sup.6 cells/kg,
about 2.1.times.10.sup.6 cells/kg, about 1.2.times.10.sup.6
cells/kg, about 2.3.times.10.sup.6 cells/kg, about
2.4.times.10.sup.6 cells/kg, about 2.5.times.10.sup.6 cells/kg,
about 2.6.times.10.sup.6 cells/kg, about 2.7.times.10.sup.6
cells/kg, about 2.8.times.10.sup.6 cells/kg, about
2.9.times.10.sup.6 cells/kg, about 3.times.10.sup.6 cells/kg, about
3.1.times.10.sup.6 cells/kg, about 3.2.times.10.sup.6 cells/kg,
about 3.3.times.10.sup.6 cells/kg, about 3.4.times.10.sup.6
cells/kg, about 3.5.times.10.sup.6 cells/kg, about
3.6.times.10.sup.6 cells/kg, about 3.7.times.10.sup.6 cells/kg,
about 3.8.times.10.sup.6 cells/kg, about 3.9.times.10.sup.6
cells/kg, about 4.times.10.sup.6 cells/kg, about 4.1.times.10.sup.6
cells/kg, about 4.2.times.10.sup.6 cells/kg, about
4.3.times.10.sup.6 cells/kg, about 4.4.times.10.sup.6 cells/kg,
about 4.5.times.10.sup.6 cells/kg, about 4.6.times.10.sup.6
cells/kg, about 4.7.times.10.sup.6 cells/kg, about
4.8.times.10.sup.6 cells/kg, about 4.9.times.10.sup.6 cells/kg, or
about 5.times.10.sup.6 cells/kg. In another embodiment, the dosage
of cells to a subject may be a single dosage or a single dosage
plus additional dosages. In other embodiments, the dosages may be
provided by way of a construct as described herein. In other
embodiments, the dosage of cells to a subject may be calculated
based on the estimated renal mass or functional renal mass.
The therapeutically effective amount of the renal cell populations
or admixtures thereof described herein can be suspended in a
pharmaceutically acceptable carrier or excipient. Such a carrier
includes, but is not limited to basal culture medium plus 1% serum
albumin, saline, buffered saline, dextrose, water, collagen,
alginate, hyaluronic acid, fibrin glue, polyethyleneglycol,
polyvinylalcohol, carboxymethylcellulose and combinations thereof.
The formulation should suit the mode of administration.
Accordingly, the invention provides a use of renal cell populations
or admixtures thereof, for example, the B2 cell population alone or
admixed with the B3 and/or B4 or B4' cell population, for the
manufacture of a medicament to treat kidney disease in a subject.
In some embodiments, the medicament further comprises recombinant
polypeptides, such as growth factors, chemokines or cytokines. In
further embodiments, the medicaments comprise a human
kidney-derived cell population. The cells used to manufacture the
medicaments can be isolated, derived, or enriched using any of the
variations provided for the methods described herein.
The renal cell preparation(s), or admixtures thereof, or
compositions are formulated in accordance with routine procedures
as a pharmaceutical composition adapted for administration to human
beings. Typically, compositions for intravenous administration,
intra-arterial administration or administration within the kidney
capsule, for example, are solutions in sterile isotonic aqueous
buffer. Where necessary, the composition can also include a local
anesthetic to ameliorate any pain at the site of the injection.
Generally, the ingredients are supplied either separately or mixed
together in unit dosage form, for example, as a cryopreserved
concentrate in a hermetically sealed container such as an ampoule
indicating the quantity of active agent. When the composition is to
be administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline.
Where the composition is administered by injection, an ampoule of
sterile water for injection or saline can be provided so that the
ingredients can be mixed prior to administration.
Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there are a wide variety of suitable formulations of pharmaceutical
compositions (see, e.g., Alfonso R Gennaro (ed), Remington: The
Science and Practice of Pharmacy, formerly Remington's
Pharmaceutical Sciences 20th ed., Lippincott, Williams &
Wilkins, 2003, incorporated herein by reference in its entirety).
The pharmaceutical compositions are generally formulated as
sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration.
One aspect of the invention further provides a pharmaceutical
formulation, comprising a renal cell preparation of the invention,
for example, the B2 cell preparation alone or in combination with
the B3 and/or B4 or B4' cell preparation, and a pharmaceutically
acceptable carrier. In some embodiments, the formulation comprises
from 10.sup.4 to 10.sup.9 mammalian kidney-derived cells.
In one aspect, the present invention provides methods of providing
one or more of the cell populations described herein, including
admixtures, to a subject in need. In one embodiment, the source of
the cell population(s) may be autologous or allogeneic, syngeneic
(autogenic or isogeneic), and any combination thereof. In instances
where the source is not autologous, the methods may include the
administration of an immunosuppressant agent. Suitable
immunosuppressant drugs include, without limitation, azathioprine,
cyclophosphamide, mizoribine, ciclosporin, tacrolimus hydrate,
chlorambucil, lobenzarit disodium, auranofin, alprostadil,
gusperimus hydrochloride, biosynsorb, muromonab, alefacept,
pentostatin, daclizumab, sirolimus, mycophenolate mofetil,
leflonomide, basiliximab, dornase .alpha., bindarid, cladribine,
pimecrolimus, ilodecakin, cedelizumab, efalizumab, everolimus,
anisperimus, gavilimomab, faralimomab, clofarabine, rapamycin,
siplizumab, saireito, LDP-03, CD4, SR-43551, SK&F-106615,
IDEC-114, IDEC-131, FTY-720, TSK-204, LF-080299, A-86281, A-802715,
GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-1011, MT-1345,
CNI-1493, CBP-2011, J-695, LJP-920, L-732531, ABX-RB2, AP-1903,
IDPS, BMS-205820, BMS-224818, CTLA4-1 g, ER-49890, ER-38925,
ISAtx-247, RDP-58, PNU-156804, LJP-1082, TMC-95A, TV-4710,
PTR-262-MG, and AGI-1096 (see U.S. Pat. No. 7,563,822). Those of
ordinary skill in the art will appreciate other suitable
immunosuppressant drugs.
The treatment methods of the subject invention involve the delivery
of an isolated renal cell population, or admixture thereof, into
individuals. In one embodiment, direct administration of cells to
the site of intended benefit is preferred. In one embodiment, the
cell preparations, or admixtures thereof, of the instant invention
are delivered to an individual in a delivery vehicle.
A subject in need may also be treated by in vivo contacting of a
native kidney with products secreted from one or more enriched
renal cell populations, and/or an admixture or construct containing
the same. The step of contacting a native kidney in vivo with
secreted products may be accomplished through the
use/administration of a population of secreted products from cell
culture media, e.g., conditioned media, or by implantation of an
enriched cell population, and admixture, or a construct capable of
secreting the products in vivo. The step of in vivo contacting
provides a regenerative effect to the native kidney.
A variety of means for administering cells and/or secreted products
to subjects will, in view of this specification, be apparent to
those of skill in the art. Such methods include injection of the
cells into a target site in a subject. Cells and/or secreted
products can be inserted into a delivery device or vehicle, which
facilitates introduction by injection or implantation into the
subjects. In certain embodiments, the delivery vehicle can include
natural materials. In certain other embodiments, the delivery
vehicle can include synthetic materials. In one embodiment, the
delivery vehicle provides a structure to mimic or appropriately fit
into the organ's architecture. In other embodiments, the delivery
vehicle is fluid-like in nature. Such delivery devices can include
tubes, e.g., catheters, for injecting cells and fluids into the
body of a recipient subject. In a preferred embodiment, the tubes
additionally have a needle, e.g., a syringe, through which the
cells of the invention can be introduced into the subject at a
desired location. In some embodiments, mammalian kidney-derived
cell populations are formulated for administration into a blood
vessel via a catheter (where the term "catheter" is intended to
include any of the various tube-like systems for delivery of
substances to a blood vessel). Alternatively, the cells can be
inserted into or onto a biomaterial or scaffold, including but not
limited to textiles, such as weaves, knits, braids, meshes, and
non-wovens, perforated films, sponges and foams, and beads, such as
solid or porous beads, microparticles, nanoparticies, and the like
(e.g., Cultispher-S gelatin beads--Sigma). The cells can be
prepared for delivery in a variety of different forms. For example,
the cells can be suspended in a solution or gel. Cells can be mixed
with a pharmaceutically acceptable carrier or diluent in which the
cells of the invention remain viable. Pharmaceutically acceptable
carriers and diluents include saline, aqueous buffer solutions,
solvents and/or dispersion media. The use of such carriers and
diluents is well known in the art. The solution is preferably
sterile and fluid, and will often be isotonic. Preferably, the
solution is stable under the conditions of manufacture and storage
and preserved against the contaminating action of microorganisms
such as bacteria and fungi through the use of, for example,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like. One of skill in the art will appreciate that the delivery
vehicle used in the delivery of the cell populations and admixtures
thereof of the instant invention can include combinations of the
above-mentioned characteristics.
Modes of administration of the isolated renal cell population(s),
for example, the B2 cell population alone or admixed with B4'
and/or B3, include, but are not limited to, systemic, intra-renal
(e.g., parenchymal), intravenous or intra-arterial injection and
injection directly into the tissue at the intended site of
activity. Additional modes of administration to be used in
accordance with the present invention include single or multiple
injection(s) via direct laparotomy, via direct laparoscopy,
transabdominal, or percutaneous. Still yet additional modes of
administration to be used in accordance with the present invention
include, for example, retrograde and ureteropelvic infusion.
Surgical means of administration include one-step procedures such
as, but not limited to, partial nephrectomy and construct
implantation, partial nephrectomy, partial pyelectomy,
vascularization with omentum+peritoneum, multifocal biopsy needle
tracks, cone or pyramidal, to cylinder, and renal pole-like
replacement, as well as two-step procedures including, for example,
organoid-internal bioreactor for replanting. In one embodiment, the
admixtures of cells are delivered via the same route at the same
time. In another embodiment, each of the cell compositions
comprising the controlled admixture are delivered separately to
specific locations or via specific methodologies, either
simultaneously or in a temporally-controlled manner, by one or more
of the methods described herein.
The appropriate cell implantation dosage in humans can be
determined from existing information relating to either the
activity of the cells, for example EPO production, or extrapolated
from dosing studies conducted in preclinical studies. From in vitro
culture and in vivo animal experiments, the amount of cells can be
quantified and used in calculating an appropriate dosage of
implanted material. Additionally, the patient can be monitored to
determine if additional implantation can be made or implanted
material reduced accordingly.
One or more other components can be added to the cell populations
and admixtures thereof of the instant invention, including selected
extracellular matrix components, such as one or more types of
collagen or hyaluronic acid known in the art, and/or growth
factors, platelet-rich plasma and drugs.
Those of ordinary skill in the art will appreciate the various
formulations and methods of administration suitable for the
secreted products described herein.
Kits
The instant invention further includes kits comprising the
polymeric matrices and scaffolds of the invention and related
materials, and/or cell culture media and instructions for use. The
instructions for use may contain, for example, instructions for
culture of the cells or administration of the cells and/or cell
products. In one embodiment, the present invention provides a kit
comprising a scaffold as described herein and instructions. In yet
another embodiment, the kit includes an agent for detection of
marker expression, reagents for use of the agent, and instructions
for use. This kit may be used for the purpose of determining the
regenerative prognosis of a native kidney in a subject following
the implantation or administration of a cell population, an
admixture, or a construct described herein. The kit may also be
used to determine the biotherapeutic efficacy of a cell population,
admixture, or construct described herein.
Reports
The methods of this invention, when practiced for commercial
purposes generally produce a report or summary of the regenerative
prognosis. The methods of this invention will produce a report
comprising a prediction of the probable course or outcome of
regeneration before and after any administration or implantation of
a cell population, an admixture, or a construct described herein.
The report may include information on any indicator pertinent to
the prognosis. The methods and reports of this invention can
further include storing the report in a database. Alternatively,
the method can further create a record in a database for the
subject and populate the record with data. In one embodiment the
report is a paper report, in another embodiment the report is an
auditory report, in another embodiment the report is an electronic
record. It is contemplated that the report is provided to a
physician and/or the patient. The receiving of the report can
further include establishing a network connection to a server
computer that includes the data and report and requesting the data
and report from the server computer. The methods provided by the
present invention may also be automated in whole or in part.
All patents, patent applications, and literature references cited
in the present specification are hereby incorporated by reference
in their entirety.
The following examples are offered for illustrative purposes only,
and are not intended to limit the scope of the present invention in
any way.
EXAMPLES
Example 1--Isolation & Characterization of Bioresponsive Renal
Cells
A case of idiopathic progressive chronic kidney disease (CKD) with
anemia in an adult male swine (Sus scrofa) provided fresh diseased
kidney tissue for the assessment of cellular composition and
characterization with direct comparison to age-matched normal swine
kidney tissue. Histological examination of the kidney tissue at the
time of harvest confirmed renal disease characterized by severe
diffuse chronic interstitial fibrosis and crescentic
glomerulonephritis with multifocal fibrosis. Clinical chemistry
confirmed azotemia (elevation of blood urea nitrogen and serum
creatinine), and mild anemia (mild reduction in hematocrit and
depressed hemoglobin levels). Cells were isolated, expanded, and
characterized from both diseased and normal kidney tissue. As shown
in FIG. 1 of Presnell et al. WO2010/056328 (incorporated herein by
reference in its entirety), a Gomori's Trichrome stain highlights
the fibrosis (blue staining indicated by arrows) in the diseased
kidney tissue compared to the normal kidney tissue. Functional
tubular cells, expressing cubulin:megalin and capable of
receptor-mediated albumin transport, were propagated from both
normal and diseased kidney tissue. Erythropoietin (EPO)-expressing
cells were also present in the cultures and were retained through
multiple passages and freeze/thaw cycles. Furthermore, molecular
analyses confirmed that the EPO-expressing cells from both normal
and diseased tissue responded to hypoxic conditions in vitro with
HIF1.alpha.-driven induction of EPO and other hypoxia-regulated
gene targets, including vEGF. Cells were isolated from the porcine
kidney tissue via enzymatic digestion with collagenase+dispase, and
were also isolated in separate experiments by performing simple
mechanical digestion and explant culture. At passage two,
explant-derived cell cultures containing epo-expressing cells were
subjected to both atmospheric (21%) and varying hypoxic (<5%)
culture conditions to determine whether exposure to hypoxia
culminated in upregulation of EPO gene expression. As noted with
rodent cultures (see Example 3), the normal pig displayed
oxygen-dependent expression and regulation of the EPO gene.
Surprisingly, despite the uremic/anemic state of the CKD pig
(Hematocrit<34, Creatinine>9.0) EPO expressing cells were
easily isolated and propagated from the tissue and expression of
the EPO gene remained hypoxia regulated, as shown in FIG. 2 of of
Presnell et al. WO/2010/056328 (incorporated herein by reference in
its entirety). As shown in FIG. 3 of Presnell et al. WO/2010/056328
(incorporated herein by reference in its entirety), cells in the
propagated cultures demonstrated the ability to self-organize into
tubule-like structures. As shown in FIG. 4 of Presnell et al.
WO/2010/056328 (incorporated herein by reference in its entirety),
the presence of functional tubular cells in the culture (at passage
3) was confirmed by observing receptor-mediated uptake of
FITC-conjugated Albumin by the cultured cells. The green dots
(indicated by thin white arrows) represent endocytosed
fluorescein-conjugated albumin which is mediated by tubular
cell-specific receptors, Megalin and Cubilin, indicating protein
reabsorption by functional tubular cells. The blue staining
(indicated by thick white arrows) is Hoescht-stained Taken
together, these data suggest that functional tubular and endocrine
cells can be isolated and propagated from porcine renal tissues,
even in renal tissues that have been severely compromised with CKD.
Furthermore, these findings support the advancement of autologous
cell-based therapeutic products for the treatment of CKD.
In addition, EPO-producing cells were isolated enzymatically from
normal adult human kidney (as described above in Example 1). As
shown in FIG. 5 of Presnell et al. WO/20101056328 (incorporated
herein by reference in its entirety), the isolation procedure
resulted in more relative EPO expression after isolation than in
the initial tissue. As shown in FIG. 6 of Presnell et al.
WO/2010/056328 (incorporated herein by reference in its entirety),
it is possible to maintain the human EPO producing cells in culture
with retention of EPO gene expression. Human cells were
cultured/propagated on plain tissue-culture treated plastic or
plastic that had been coated with some extracellular matrix, such
as, for instance, fibronectin or collagen, and all were found to
support EPO expression over time.
Example 2--Isolation & Enrichment of Specific Bioreactive Renal
Cells
Kidney cell isolation: Briefly, batches of 10, 2-week-old male
Lewis rat kidneys were obtained from a commercial supplier (Hilltop
Lab Animals Inc.) and shipped overnight in Viaspan preservation
medium at a temperature around 4.degree. C. All steps described
herein were carried out in a biological safety cabinet (BSC) to
preserve sterility. The kidneys were washed in Hank's balanced salt
solution (HBSS) 3 times to rinse out the Viaspan preservation
medium. After the third wash the remaining kidney capsules were
removed as well as any remaining stromal tissue. The major calyx
was also removed using micro dissection techniques. The kidneys
were then finely minced into a slurry using a sterile scalpel. The
slurry was then transferred into a 50 ml conical centrifuge tube
and weighed. A small sample was collected for RNA and placed into
an RNAse-free sterile 1.5 ml micro-centrifuge tube and snap frozen
in liquid nitrogen. Once frozen, it was then transferred to the -80
degree freezer until analysis. The tissue weight of 10 juvenile
kidneys equaled approximately 1 gram. Based on the weight of the
batch, the digestion medium was adjusted to deliver 20 mls of
digestion medium per 1 gram of tissue. Digestion buffer for this
procedure contained 4 Units of Dispase 1(Stem Cell Tech) in HBSS,
300 Units/ml of Collagenase type IV (Worthington) with 5 mM
CaCl.sub.2 (Sigma).
The appropriate volume of pre-warmed digestion buffer was added to
the tube, which was then sealed and placed on a rocker in a
37.degree. C. incubator for 20 minutes. This first digestion step
removes many red blood cells and enhances the digestion of the
remaining tissue. After 20 minutes, the tube was removed and placed
in the BSC. The tissue was allowed to settle at the bottom of the
tube and then the supernatant was removed. The remaining tissue was
then supplemented with fresh digestion buffer equaling the starting
volume. The tube was again placed on a rocker in a 37.degree. C.
incubator for an additional 30 minutes.
After 30 minutes the digestion mixture was pipetted through a 70 nm
cell strainer (BD Falcon) into an equal volume of neutralization
buffer (DMEM w/10% FBS) to stop the digestion reaction. The cell
suspension was then washed by centrifugation at 300.times.g for 5
min. After centrifugation, the pellet was then re-suspended in 20
mls KSFM medium and a sample acquired for cell counting and
viability assessment using trypan blue exclusion. Once the cell
count was calculated, 1 million cells were collected for RNA,
washed in PBS, and snap frozen in liquid nitrogen. The remaining
cell suspension was brought up to 50 mls with KSFM medium and
washed again by centrifugation at 300.times.g for 5 minutes. After
washing, the cell pellet was re-suspended in a concentration of 15
million cells per ml of KSFM.
Five milliliters of kidney cell suspension were then added to 5 mls
of 30% (w/v) Optiprep.RTM. in 15 ml conical centrifuge tubes (BD
Falcon) and mixed by inversion 6 times. This formed a final mixture
of 15% (w/v) of Optiprep.RTM.. Post inversion, tubes were carefully
layered with 1 mL PBS. The tubes were centrifuged at 800.times.g
for 15 minutes without brake. After centrifugation, the tubes were
removed and a cell band was formed at the top of the mixing
gradient. There was also a pellet containing red blood cells, dead
cells, and a small population of live cells that included some
small less granular cells, some epo-producing cells, some tubular
cells, and some endothelial cells. The band was carefully removed
using a pipette and transferred to another 15 ml conical tube. The
gradient medium was removed by aspiration and the pellet was
collected by re-suspension in 1 ml KSFM. The band cells and pellet
cells were then recombined and re-suspended in at least 3 dilutions
of the collected band volume using KSFM and washed by
centrifugation at 300.times.g for 5 minutes. Post washing, the
cells were re-suspended in 20 mls of KSFM and a sample for cell
counting was collected. Once the cell count was calculated using
trypan blue exclusion, 1 million cells were collected for an RNA
sample, washed in PBS, and snap frozen in liquid nitrogen.
Pre-Culture `Clean-up` to Enhance Viability and Culture Performance
of Specific Bioactive Renal Cells Using Density Gradient
Separation: To yield a clean, viable population of cells for
culture, a cell suspension was first generated as described above
in "Kidney Cell isolation". As an optional step and as a means of
cleaning up the initial preparation, up to 100 million total cells,
suspended in sterile isotonic buffer were mixed thoroughly 1:1 with
an equal volume of 30% Optiprep.RTM. prepared at room temperature
from stock 60% (w/v) iodixanol (thus yielding a final 15% w/v
Optiprep solution) and mixed thoroughly by inversion six times.
After mixing, Intl PBS buffer was carefully layered on top of the
mixed cell suspension. The gradient tubes were then carefully
loaded into the centrifuge, ensuring appropriate balance. The
gradient tubes were centrifuged at 800.times.g for 15 minutes at
25.degree. C. without brake. The cleaned-up cell population
(containing viable and functional collecting duct, tubular,
endocrine, glomerular, and vascular cells) segmented between 6% and
8% (w/v) Optiprep.RTM., corresponding to a density between
1.025-1.045 g/mL. Other cells and debris pelleted to the bottom of
the tube.
Kidney Cell Culture: The combined cell band and pellet were then
plated in tissue culture treated triple flasks (Nunc T500) or
equivalent at a cell concentration of 30,000 cells per cm2 in 150
mls of a 50:50 mixture of DMEM(high glucose)/KSFM containing 5%
(v/v) FBS, 2.5 .mu.g EGF, 25 mg BPE, 1.times.ITS
(insulin/transferrin/sodium selenite medium supplement) with
antibiotic/antimycotic. The cells were cultured in a humidified 5%
CO2 incubator for 2-3 days, providing a 21% atmospheric oxygen
level for the cells. After two days, the medium was changed and the
cultures were placed in 2% oxygen-level environment provided by a
CO2/Nitrogen gas multigas humidified incubator (Sanyo) for 24 hrs.
Following the 24 hr incubation, the cells were washed with 60 mls
of 1.times.PBS and then removed using 40 mls 0.25% (w/v)
trypsin/EDTA (Gibco). Upon removal, the cell suspension was
neutralized with an equal volume of KSFM containing 10% FBS. The
cells were then washed by centrifugation 300.times.g for 10
minutes. After washing, the cells were re-suspended in 20 mls of
KSFM and transferred to a 50 ml conical tube and a sample was
collected for cell counting. Once the viable cell count was
determined using trepan blue exclusion, 1 million cells were
collected for an RNA sample, washed in PBS, and snap frozen in
liquid nitrogen. The cells were washed again in PBS and collected
by centrifugation at 300.times.g for 5 minutes. The washed cell
pellet was re-suspended in KSFM at a concentration of 37.5 million
cells/ml.
Enriching for Specific Bioactive Renal Cells Using Density Step
Gradient Separation: Cultured kidney cells, predominantly composed
of renal tubular cells but containing small subpopulations of other
cell types (collecting duct, glomerular, vascular, and endocrine)
were separated into their component subpopulations using a density
step gradient made from multiple concentrations w/v of iodixanol
(Optiprep). The cultures were placed into a hypoxic environment for
up to 24 hours prior to harvest and application to the gradient. A
stepped gradient was created by layering four different density
mediums on top of each other in a sterile 15 mL conical tube,
placing the solution with the highest density on the bottom and
layering to the least dense solution on the top. Cells were applied
to the top of the step gradient and centrifuged, which resulted in
segregation of the population into multiple bands based on size and
granularity.
Briefly, densities of 7, 11, 13, and 16% Optiprep.RTM. (60% w/v
Iodixanol) were made using KFSM medium as diluents. For example:
for 50 mls of 7% (w/v) Optiprep.RTM., 5.83 mls of stock 60% (w/v)
Iodixanol was added to 44.17 mls of KSFM medium and mixed well by
inversion. A peristaltic pump (Master Flex L/S) loaded with sterile
L/S 16 Tygon tubing connected to sterile capillary tubes was set to
a flow rate of 2 ml per minute, and 2 mL of each of the four
solutions was loaded into a sterile conical 15 mL tube, beginning
with the 16% solution, followed by the 13% solution, the 11%
solution, and the 7% solution. Finally, 2 mL of cell suspension
containing 75 million cultured rodent kidney cells was loaded atop
the step gradient (suspensions having been generated as described
above in `Kidney cell Culture`). Importantly, as the pump was
started to deliver the gradient solutions to the tube, care was
taken to allow the fluid to flow slowly down the side of the tube
at a 45.degree. angle to insure that a proper interface formed
between each layer of the gradient. The step gradients, loaded with
cells, were then centrifuged at 800.times.g for 20 minutes without
brake. After centrifugation, the tubes were carefully removed so as
not to disturb each interface. Five distinct cell fractions
resulted (4 bands and a pellet) (B1-B4, +Pellet) (see FIG. 1A, left
conical tube). Each fraction was collected using either a sterile
disposable bulb pipette or a 5 ml pipette and characterized
phenotypically and functionally (See Example 10 of Presnell et al.
WO/2010/056328). When rodent kidney cell suspensions are subjected
to step-gradient fractionation immediately after isolation, the
fraction enriched for tubular cells (and containing some cells from
the collecting duct) segments to a density between 1.062-1.088
g/mL. In contrast, when density gradient separation was performed
after ex viva culture, the fraction enriched for tubular cells (and
containing some cells from the collecting duct) segmented to a
density between 1.051-1.062 g/mL. Similarly, when rodent kidney
cell suspensions are subjected to step-gradient fractionation
immediately after isolation, the fraction enriched for
epo-producing cells, glomerular podocytes, and vascular cells
("B4") segregates at a density between 1.025-1.035 g/mL. In
contrast, when density gradient separation was performed after ex
vivo culture, the fraction enriched for epo-producing cells,
glomerular podocytes, and vascular cells ("B4") segregated at a
density between 1.073-1.091 g/mL. Importantly, the post-culture
distribution of cells into both the "B2" and the "B4" fractions was
enhanced by exposure (for a period of about 1 hour to a period of
about 24 hours) of the cultures to a hypoxic culture environment
(hypoxia being defined as <21% (atmospheric) oxygen levels prior
to harvest and step-gradient procedures (additional details
regarding hypoxia-effects on band distribution are provided in
Example 3).
Each band was washed by diluting with 3.times. the volume of KSFM,
mixed well, and centrifuged for 5 minutes at 300.times.g. Pellets
were re-suspended in 2 mls of KSFM and viable cells were counted
using trypan blue exclusion and a hemacytometer. 1 million cells
were collected for an RNA sample, washed in PBS, and snap frozen in
liquid nitrogen. The cells from B2 and B4 were used for
transplantation studies into uremic and anemic female rats,
generated via a two-step 5/6 nephrectomy procedure at Charles River
Laboratories. Characteristics of B4 were confirmed by quantitative
real-time PCR, including oxygen-regulated expression of
erythropoietin and vEGF, expression of glomerular markers (nephrin,
podocin), and expression of vascular markers (PECAM). Phenotype of
the `B2` fraction was confirmed via expression of E-Cadherin,
N-Cadherin, and Aquaporin-2. See FIGS. 49A and 49B of Presnell et
al. WO/2010/056328.
Thus, use of the step gradient strategy allows not only the
enrichment for a rare population of epo-producing cells (B4), but
also a means to generate relatively enriched fractions of
functional tubular cells (B2) (see FIGS. 50 & 51 of Presnell et
al. WO/2010/056328). The step gradient strategy also allows
EPO-producing and tubular cells to be separated from red blood
cells, cellular debris, and other potentially undesirable cell
types, such as large cell aggregates and certain types of immune
cells.
The step gradient procedure may require tuning with regard to
specific densities employed to provide good separation of cellular
components. The preferred approach to tuning the gradient involves
1) running a continuous density gradient where from a high density
at the bottom of the gradient (16-21% Optiprep, for example) to a
relatively low density at the top of the gradient (5-10%, for
example). Continuous gradients can be prepared with any standard
density gradient solution (Ficoll, Percoll, Sucrose, iodixanol)
according to standard methods (Axis Shield). Cells of interest are
loaded onto the continuous gradient and centrifuged at 800.times.G
for 20 minutes without brake. Cells of similar size and granularity
tend to segregate together in the gradients, such that the relative
position in the gradient can be measured, and the specific gravity
of the solution at that position also measured. Thus, subsequently,
a defined step gradient can be derived that focuses isolation of
particular cell populations based on their ability to transverse
the density gradient under specific conditions. Such optimization
may need to be employed when isolating cells from unhealthy vs.
healthy tissue, or when isolating specific cells from different
species. For example, optimization was conducted on both canine and
human renal cell cultures, to insure that the specific B2 and B4
subpopulations that were identified in the rat were isolatable from
the other species. The optimal gradient for isolation of rodent B2
and B4 subpopulations consists of (w/v) of 7%, 11%, 13%, and 16%
Optiprep. The optimal gradient for isolation of canine B2 and B4
subpopulations consists of (w/v) of 7%, 10%, 11%, and 16% Optiprep.
The optimal gradient for isolation of human B2 and B4
subpopulations consists of (w/v) 7%, 9%, 11%, 16%. Thus, the
density range for localization of B2 and B4 from cultured rodent,
canine, and human renal cells is provided in Table 2.1.
TABLE-US-00001 TABLE 2.1 Species Density Ranges. Step Gradient
Species Density Ranges g/ml Band Rodent Canine Human B2 1.045-1.063
g/ml 1.045-1.058 g/ml 1.045-1.052 g/ml B4 1.073-1.091 g/ml
1.063-1.091 g/ml 1.063-1.091 g/ml
Example 3--Low-Oxygen Culture Prior to Gradient Affects Bland
Distribution, Composition, and Gene Expression
To determine the effect of oxygen conditions on distribution and
composition of prototypes B2 and B4, neokidney cell preparations
from different species were exposed to different oxygen conditions
prior to the gradient step. A rodent neo-kidney augmentation (NKA)
cell preparation (RK069) was established using standard procedures
for rat cell isolation and culture initiation, as described supra.
All flasks were cultured for 2-3 days in 21% (atmospheric) oxygen
conditions. Media was changed and half of the flasks were then
relocated to an oxygen-controlled incubator set to 2% oxygen, while
the remaining flasks were kept at the 21% oxygen conditions, for an
additional 24 hours. Cells were then harvested from each set of
conditions using standard enzymatic harvesting procedures described
supra. Step gradients were prepared according to standard
procedures and the "normoxic" (21% oxygen) and "hypoxic" (2%
oxygen) cultures were harvested separately and applied side-by-side
to identical step gradients. (FIG. 2). While 4 bands and a pellet
were generated in both conditions, the distribution of the cells
throughout the gradient was different in 21% and 2% oxygen-cultured
batches (Table 1). Specifically, the yield of B2 was increased with
hypoxia, with a concomitant decrease in B3. Furthermore, the
expression of B4-specific genes (such as erythropoietin) was
enhanced in the resulting gradient generated from the
hypoxic-cultured cells (FIG. 73 of Presnell et al.
WO/2010/056328).
A canine NKA cell preparation (DK008) was established using
standard procedures for dog cell isolation and culture (analogous
to rodent isolation and culture procedures), as described supra.
All flasks were cultured for 4 days in 21% (atmospheric) oxygen
conditions, then a subset of flasks were transferred to hypoxia
(2%) for 24 hours while a subset of the flasks were maintained at
21%. Subsequently, each set of flasks was harvested and subjected
to identical step gradients (FIG. 3). Similar to the rat results
(Example 1), the hypoxic-cultured dog cells distributed throughout
the gradient differently than the atmospheric oxygen-cultured dog
cells (Table 3.1). Again, the yield of B2 was increased with
hypoxic exposure prior to gradient, along with a concomitant
decrease in distribution into B3.
TABLE-US-00002 TABLE 3.1 Rat (RK069) Dog (DK008) 2% O2 21% O2 2% O2
21% O2 B1 0.77% 0.24% 1.20% 0.70% B2 88.50% 79.90% 64.80% 36.70% B3
10.50% 19.80% 29.10% 40.20% B4 0.23% 0.17% 4.40% 21.90%
The above data show that pre-gradient exposure to hypoxia enhances
composition of B2 as well as the distribution of specific
specialized cells (erythropoietin-producing cells, vascular cells,
and glomerular cells) into B4. Thus, hypoxic culture, followed by
density-gradient separation as described supra, is an effective way
to generate `B2` and `B4` cell populations, across species.
Example 4--Isolation of Tubular/Glomerular Cells from Human
Kidney
Tubular and glomerular cells were isolated and propagated from
normal human kidney tissue by the enzymatic isolation methods
described throughout. By the gradient method described above, the
tubular cell fraction was enriched ex vivo and after culture. As
shown in FIG. 68 of of Presnell et al. WO/2010/056328 (incorporated
herein by reference in its entirety), phenotypic attributes were
maintained in isolation and propagation. Tubular cell function,
assessed via uptake of labeled albumin, was also retained after
repeated passage and cryopreservation. FIG. 69 of Presnell et al.
WO/2010/056328 (incorporated herein by reference in its entirety)
shows that when tubular-enriched and tubular-depleted populations
were cultured in 3D dynamic culture, a marked increase in
expression of tubular marker, cadherin, was expressed in the
tubular-enriched population. This confirms that the enrichment of
tubular cells can be maintained beyond the initial enrichment when
the cells are cultured in a 3D dynamic environment.
Example 5--Further Separation of EPO-Producing Cells via Flow
Cytometry
The same cultured population of kidney cells described above in
Example 2 was subjected to flow cytometric analysis to examine
forward scatter and side scatter. The small, less granular
EPO-producing cell population was discernable (8.15%) and was
separated via positive selection of the small, less granular
population using the sorting capability of a flow cytometer (see
FIG. 70 of Presnell et al. WO/2010/056328 (incorporated herein by
reference in its entirety)).
Example 6--Characterisation of an Unfractionated Mixture of Renal
Cells Isolated from an Autoimmune Glomerulonephritis Patient
Sample
An unfractionated mixture of renal cells was isolated, as described
above, from an autoimmune glomerulonephritis patient sample. To
determine the unbiased genotypic composition of specific
subpopulations of renal cells isolated and expanded from kidney
tissue, quantitative real time PCR (qrtpcr) analysis (Brunskill et
al., supra 2008) was employed to identify differential
cell-type-specific and pathway-specific gene expression patterns
among the cell subtractions. As shown in Table 6.1. HK20 is an
autoimmune glomerulonephritis patient sample. Table 6.2 shows that
cells generated from HK20 are lacking glomerular cells, as
determined by qRTPCR.
Example 7--Genetic Profiling of Therapeutically Relevant Renal
Bioactive Cell Populations Isolated from a Case of Focal Segmental
Glomerulosclerosis
To determine the unbiased genotypic composition of specific
subpopulations of renal cells isolated and expanded from kidney
tissue, quantitative real time PCR (griper) analysis (Brunskill et
al., supra 2008) was employed to identify differential
cell-type-specific and pathway-specific gene expression patterns
among the cell subtractions. Human preparation HK023, derived from
a case of focal segmental glomerulosclerosis (FSGS) in which a
large portion of glomeruli had been destroyed, was evaluated for
presence of glomerular cells in the B4 fraction at the time of
harvest. In brief, unfractionated (UNFX) cultures were generated
(Aboushwareb et al., supra 2008) and maintained independently from
each of (4) core biopsies taken from the kidney using standard
biopsy procedures. After (2) passages of UNFX ex vivo, cells were
harvested and subjected to density gradient methods (as in Example
8) to generate subtractions, including subtraction B4, which is
known to be enriched for endocrine, vascular, and glomerular cells
based on work conducted in rodent, dog, and other human
specimens.
TABLE-US-00003 TABLE 6.1 Cause of Death (D) Creatinine Etiology of
or Kidney Removal BUN sCreat Clearance HCT NB sPHOS Key
Histopathologic Sample ID Species Age/Gender Renal Disease (KR)
(mg/dL) (mg/dL) (CC)/GFR/eGFR (%) (mg/dL) (mg/DL) uPRO Featu- res
PK001 Swine >1 yr/M Idiopathic (D) Renal 75 9.5 na 34.1 10.6 6.3
na marked fibrosis; nephropathy Failure glomerular hypertrophy with
focal sclerosis; tubular dilatation with protein casts PK002 Swine
>1 yr/M no renal disease (D) na na na na na na na normal kidney
Sacrifice histology DK001 Canine >11 yr/M age-related renal (D)
24 1/1 ma 40.1 13.5 6.6 0 diffuse degeneration Sacrifice glomerular
with fatty lipidosis with metaplasia of focal segmental flomeruli
glomerular sclerosis DK002 Canine >2 yr/M chronic (D) 20 0.8 na
47 15.9 3.6 >3.0 chronic glomerulonephritis Sacrifice
glomerulonephritis with chronic inflammation, glomerular sclerosis,
and moderate fibrosis HK016 Human 2 mo/F no renal disease (D) Head
13 0.4 na 26.6 9.6 8.6 trace normal neonatal Trauma kidney
histology HK017 Human 35 yr/F Petechial (D) CVA 12 2.9 na 26 8.8
6.3 trace normal tubular hemorrhage histology; no secondary to DIC
fibrosis; fibrin thrombi throughout glomerular capillaries HK018
Human 48 yr/F secondary to (D) CV/Renal 40 8.6 8.06 (CC) 24.6 8.1
6.7 na marked fibrosis; hypertension, Failure (anuric) glomerular
NIDDM, and sclerosis; tubular heart disease dilatation with protein
casts HK019 Human 52 yr/F secondary to (D) CV/Renal 127 5.7 14.5
(CC) 23.7 8.4 12.4 >300 diffuse moderate hypertension, Failure
glomerular NIDDM, and obsolescence heart disease with thickening of
Bowman's capsule; peri- glomerulas fibrosis; moderate tubular
injury with diffuse tubulo-interstitial fibrosis, tubular
dilatation with protein casts. HK020 Human 54 yr/F auto-immune (D)
CV/Stroke 94 16.6 4.35 (CC) 29 9.6 5.4 na Severe end-
glomerulonephritis (anuric) stage renal disease; no functional
glomeruli observed; severe glomerular sclerosis and interstitial
fibrosis with chronic inflammation, tubular congestion with protein
casts. HK021 Human 15 mo/M no renal disease (D) trauma 11 0.4 73.4
(CC) 29 10.3 3.4 trace normal kidney histology HK022 Human 60 yr/M
secondary to (D) CVA/ 53 3.3 17 (GFR) 31.1 10 1.8 100 Severe end-
hypertension Intracranial stage renal NIDDM, and hemorrhage
disease; diffuse heart disease severe glomerulosclerosis;
interstitial fibrosis and tubular atrophy with protein casts. HK023
Human 18 yr/M focal segmental (KR) failed 28 6.4 13.8 (GFR) 36 11.8
6.4 na focal segmental glomerulosclerosis, kidneys
glomerulosclerosis nephrotic removed (10-15% of syndrome, prior to
glomerufisclerosed), hypertension transplant associated with
diffuse mesangial hypercellularity; diffuse, focally accentuated
moderate to marked interstitial fibrosis and tubular atrophy;
marked chronic active interstitial nephritis CKD Rats Rat 4-6 mo/F
renal mass (D) Renal 96.5 .+-. 14* 2.4 .+-. 0.2* 0.48 0.48 .+-.
0.3* 39.3 .+-. 1.8* 13.2 .+-. 0.6* 10.2 .+-. 1.2* 1420 .+-. 535*
interstitial (5/6Nx) (Lewis) insufficiency Failure (eGFR) fibrosis;
n = 16 glomerular atrophy and sclerosis; tubular degeneration and
dilatation Healthy Rat 4-6 mo/F None (D) 16.9 .+-. 0.6* 0.4 .+-.
0.02* 1.7 .+-. 0.1* 46.1 .+-. 0.6* 14.7 .+-. 0.3* 6.8 .+-. 0.3* 36
.+-. 13* normal adult rats (age- (Lewis) Sacrifice (eGFR) kidney
histology matched; n = 16) Diabetic Rat 9 mo/M obesity, diabetes
(D) 30.9 .+-. 4.8* 0.6 .+-. 0.5* 3.8 .+-. 0.3* na na 5.3 .+-. 0.4*
931 .+-. 0.4* arteriolar Nephropathy (ZSF1) Sacrifice (eGFR)
thickening, Rats severe tubular (Ob/ObZSF1); degeneration, n = 10
dilation, and atrophy, and protein casts in the Bowman's space and
tubular lumens (REF: Prabhakar, 2007 jASN); by 20 weeks of age Lean
ZSF1 Rat 9 mo/M None (D) 18.9 .+-. 2.9* 0.4 .+-. 0.05* 6.4 .+-.
1.2* na na 4.6 .+-. 0.5* 296 .+-. 69* moderate Rats (Age- (ZSF1)
Sacrifice (eGFR) arteriolar Matched); thickening; n = 10 normal
tubular and glomerular structures (REF: Prabhakar 2007 JASN); at 20
weeks of age
TABLE-US-00004 TABLE 6.2 Compartmental analysis of cultured human,
swine, and rat renal cells. Sample TUBULAR GLOMERULAR DUCTULAR
OTHER ID E-CAD N-CAD AQP-1 CUB CYP24 ALB-U NEPH PODO AQP-2 EPO vEGF
KDR CD31 SSC- /FSC PK001 + nd nd nd nd ++ nd nd nd +R + nd nd +
PK002 + nd nd nd nd + nd nd nd +R + nd nd + HK016 3.03 0.83 0.0001
0.0006 0.055 + 0.0004 0.0050 0.0001 0.020R 0.85 0.0- 01 trace +
HK017 0.66 0.83 0.0009 0.0002 0.046 ++ trace 0.0001 0.0003 0.032R
0.36 0.0- 02 0.0003 + HK018 0.61 1.59 0.0001 0.0003 0.059 + 0.0002
- - 0.004R 0.36 0.003 trace +- HK019 0.62 2.19 0.026 0.0008 0.068
+/- 0.0009 0.0003 0.0020 0.076R 0.40 0.002 0.0040 + HK020 0.07 1.65
0.0003 0.0007 0.060 +++ - - - 0.011R 0.40 0.002 - + Healthy + + + +
+ + + + + +R + + + + Lewis Rat (male) Rat CKD + + + + nd nd + + nd
+R nd nd nd + model (5/6 NX Lewis)
The B4 fractions were collected separately from each independent
UNFX sample of HK023, appearing as distinct bands of cells with
buoyant density between 1.063-1.091 g/mL. RNA was isolated from
each sample and examined for expression of Podocin (glomerular cell
marker) and PECAM (endothelial cell marker) by quantitative
real-time PCR. As expected from a biopsy-generated sample from a
case of severe FSGS, the presence of podocin(+) glomerular cells in
B4 fractions was inconsistent, with podocin undetectable in 2/4 of
the samples. In contrast, PECAM+ vascular cells were consistently
present in the B4 fractions of 4/4 of the biopsy-initiated
cultures. Thus, the B4 fraction can be isolated at the 1.063-1.091
g/mL density range, even from human kidneys with severe disease
states.
TABLE-US-00005 TABLE 7.1 Expression of Podocin and PECAM for
detection of glomerular and vascular cells in subfraction B4
isolated from a case of FSGS. HK023/ RQ RQ Biopsy (Podocin)/B4
(PECAM)/B4 #1/p2 0.188 0.003 #2/p2 ND 0.02 #3/p2 40.1 0.001 #4/p2
ND 0.003
Further, as shown in Table 7.2, human sample (HK018) displayed
undetected Podocin (glomerular marker) by qRTPCR after density
gradient centrifugation.
TABLE-US-00006 TABLE 7.2 HK018 Post-Gradient gene expression
characterization of B2 & B4' Gene RQ(Unfx) RQ(B2) RQ(B4) B2/B4
Podocin 1 ND ND -- VegF 1 1.43 1.62 0.9 Aqp1 1 1.7 1.2 1.4 Epo 1
0.9 0.5 1.8 Cubilin 1 1.2 0.7 1.7 Cyp 1 1.2 1.4 0.85 Ecad 1 1.15
0.5 2.3 Ncad 1 1.02 0.72 1.4
Example 8--Enrichment/Depletion of Viable Kidney Cell Types Using
Fluorescent Activated Cell Sorting (FACS)
One or more isolated kidney cells may be enriched, and/or one or
more specific kidney cell types may be depleted from isolated
primary kidney tissue using fluorescent activated cell sorting
(FACS). REAGENTS: 70% ethanol; Wash buffer (PBS); 50:50 Kidney cell
medium (50% DMEM high glucose): 50% Keratinocyte-SFM, Trypan Blue
0.4%; Primary antibodies to target kidney cell population such as
CD31 for kidney endothelial cells and Nephrin for kidney glomerular
cells. Matched isotype specific fluorescent secondary antibodies;
Staining buffer (0.05% BSA in PBS) PROCEDURE: Following standard
procedures for cleaning the biological safety cabinet (BSC), a
single cell suspension of kidney cells from either primary
isolation or cultured cells may be obtained from a T500 T/C treated
flask and resuspend in kidney cell medium and place on ice. Cell
count and viability is then determined using trypan blue exclusion
method. For kidney cell enrichment/depletion of, for example,
glomerular cells or endothelial cells from a heterogeneous
population, between 10 and 50e6 live cells with a viability of at
least 70% are obtained. The heterogeneous population of kidney
cells is then stained with primary antibody specific for target
cell type at a starting concentration of 1 .mu.g/0.1 ml of staining
buffer/1.times.10.sup.6 cells (titer if necessary). Target antibody
can be conjugated such as CD31 PE (specific for kidney endothelial
cells) or un-conjugated such as Nephrin (specific for kidney
glomerular cells).
Cells are then stained for 30 minutes on ice or at 4.degree. C.
protected from light. After 30 minutes of incubation, cells are
washed by centrifugation at 300.times.g for 5 min. The pellet is
then resuspended in either PBS or staining buffer depending on
whether a conjugated isotype specific secondary antibody is
required. If cells are labeled with a fluorochrome conjugated
primary antibody, cells are resuspended in 2 mls of PBS per 10e7
cells and proceed to FACS aria or equivalent cell sorter. If cells
are not labeled with a fluorochrome conjugated antibody, then cells
are labeled with an isotype specific fluorochrome conjugated
secondary antibody at a starting concentration of 1 .mu.g/0.1
ml/1e6 cells.
Cells are then stained for 30 min. on ice or at 4.degree. C.
protected from light. After 30 minutes of incubation, cells are
washed by centrifugation at 300.times.g for 5 min. After
centrifugation, the pellet is resuspended in PBS at a concentration
of 5e6/ml of PBS and then 4 mls per 12.times.75 mm is transferred
to a sterile tube.
FACs Aria is prepared for live cell sterile sorting per
manufacturer's instructions (BD FACs Aria User Manual). The sample
tube is loaded into the FACs Aria and PMT voltages are adjusted
after acquisition begins. The gates are drawn to select kidney
specific cells types using fluorescent intensity using a specific
wavelength. Another gate is drawn to select the negative
population. Once the desired gates have been drawn to encapsulate
the positive target population and the negative population, the
cells are sorted using manufacturer's instructions.
The positive target population is collected in one 15 ml conical
tube and the negative population in another 15 ml conical tube
filled with 1 ml of kidney cell medium. After collection, a sample
from each tube is analyzed by flow cytometry to determine purity.
Collected cells are washed by centrifugation at 300.times.g for 5
min. and the pellet is resuspended in kidney cell medium for
further analysis and experimentation.
Example 9--Enrichment/Depletion of Kidney Cell Types Using Magnetic
Cell Sorting
One or more isolated kidney cells may be enriched and/or one or
more specific kidney cell types may be depleted from isolated
primary kidney tissue. REAGENTS: 70% ethanol, Wash buffer (PBS),
50:50 Kidney cell medium (50% DMEM high glucose): 50%
Keratinocyte-SFM, Trypan Blue 0.4%, Running Buffer(PBS, 2 mM EDTA,
0.5% BSA), Rinsing Buffer (PBS, 2 mM EDTA). Cleaning Solution (70%
v/v ethanol), FCR Blocking reagent, Miltenyi microbeads specific
for either IgG isotype, target antibody such as CD31(PECAM) or
Nephrin, or secondary antibody. PROCEDURE: Following standard
procedures for cleaning the biological safety cabinet (BSC), a
single cell suspension of kidney cells from either primary
isolation or culture is obtained and resuspended in kidney cell
medium. Cell count and viability is determined using trypan blue
exclusion method.
For kidney cell enrichment/depletion of, for example, glomerular
cells or endothelial cells from a heterogeneous population, at
least 10e6 up to 4e9 live cells with a viability of at least 70% is
obtained.
The best separation for enrichment/depletion approach is determined
based on target cell of interest. For enrichment of a target
frequency of less than 10%, for example, glomerular cells using
Nephrin antibody, the Miltenyi autoMACS, or equivalent, instrument
program POSSELDS (double positive selection in sensitive mode) is
used. For depletion of a target frequency of greater than 10%, the
Miltenyi autoMACS, or equivalent, instrument program DEPLETES
(depletion in sensitive mode) is used.
Live cells are labeled with target specific primary antibody, for
example, Nephrin rb polyclonal antibody for glomerular cells, by
adding 1 .mu.g/10e6 cells/0.1 ml of BSA with 0.05% BSA in a 15 ml
conical centrifuge tube, followed by incubation for 15 minutes at
4.degree. C.
After labeling, cells are washed to remove unbound primary antibody
by adding 1-2 ml of buffer per 10e7 cells followed by
centrifugation at 300.times.g for 5 min. After washing, isotype
specific secondary antibody, such as chicken anti-rabbit PE at 1
.mu.g/10e6/0.1 ml of BSA with 0.05% BSA, is added, followed by
incubation for 15 minutes at 4.degree. C.
After incubation, cells are washed to remove unbound secondary
antibody by adding 1-2 ml of buffer per 10e7 cells followed by
centrifugation at 300.times.g for 5 min. The supernatant is
removed, and the cell pellet is resuspended in 60 .mu.l of buffer
per 10e7 total cells followed by addition of 20 .mu.l of FCR
blocking reagent per 10e7 total cells, which is then mixed well.
Add 20 .mu.l of direct MACS microbeads (such as anti-PE microbeads)
and mix and then incubate for 15 min at 4.degree. C.
After incubation, cells are washed by adding 10-20.times. the
labeling volume of buffer and centrifuging the cell suspension at
300.times.g for 5 min. and resuspending the cell pellet in 500
.mu.l-2 mls of buffer per 10e8 cells.
Per manufacturer's instructions, the autoMACS system is cleaned and
primed in preparation for magnetic cell separation using autoMACS.
New sterile collection tubes are placed under the outlet ports. The
autoMACS cell separation program is chosen. For selection the
POSSELDS program is chosen. For depletion the DEPLETES program is
chosen.
The labeled cells are inserted at uptake port, then beginning the
program. After cell selection or depletion, samples are collected
and placed on ice until use. Purity of the depleted or selected
sample is verified by flow cytometry.
Example 10--Cells with Therapeutic Potential Can be Isolated and
Propagated from Normal and Chronically-Diseased Kidney Tissue
The objective of the present study was to determine the functional
characterization of human NKA cells through high content analysis
(HCA). High-content imaging (HCI) provides simultaneous imaging of
multiple sub-cellular events using two or more fluorescent probes
(multiplexing) across a number of samples. High-content Analysis
(HCA) provides simultaneous quantitative measurement of multiple
cellular parameters captured in High-Content Images. In brief,
unfractionated (UNFX) cultures were generated (Aboushwareb et al.,
supra 2008) and maintained independently from core biopsies taken
from five human kidneys with advanced chronic kidney disease (CKD)
and three non-CKD human kidneys using standard biopsy procedures.
After (2) passages of UNTX ex vivo, cells were harvested and
subjected to density gradient methods (as in Example 2) to generate
subfractions, including subfractions B2, B3, and/or B4.
Human kidney tissues were procured from non-CKD and CKD human
donors as summarized in Table 10.1. FIG. 4 shows histopathologic
features of the HK17 and HK19 samples. Ex vivo cultures were
established from all non-CKD (3/3) and CKD (5/5) kidneys. High
content analysis (HCA) of albumin transport in human NKA cells
defining regions of interest (ROI) is shown in FIG. 5 (HCA of
albumin transport in human NKA cells). Quantitative comparison of
albumin transport in NKA cells derived from non-CKD and CKD kidney
is shown in FIG. 6. As shown in FIG. 6, albumin transport is not
compromised in CKD-derived NKA cultures. Comparative analysis of
marker expression between tubular-enriched B2 and tubular
cell-depleted B4 subfractions is shown in FIG. 7 (CK8/18/19).
TABLE-US-00007 TABLE 10.1 Cause of Creatinine Death (D) Clearance
Etiology or Kidney BUN (CC)/ HB sPHOS Sample Age/ of Renal Removal
(mg/ sCREAT GFR/ HCT (mg/ (mg/ Key Histopathologic ID Gender
Disease (KR) dL) (mg/dL) eGFR (%) dL) dL) uPRO Features HK016 2
mo/F no renal (D) Trauma 13 0.4 na 26.6 9.6 8.6 trace normal
neonatal kidney disease histology HK017 35 yr/F Petechial (D) CVA
12 2.9 na 26 8.8 6.3 trace normal tubular histology; no hemorrhage
fibrosis; fibrin thrombi secondary throughout glomerular to DIC
capillaries HK018 43 yr/F secondary to (D) CV/ 40 8.6 8.06 (CC)
24.6 8.1 6.7 na marked fibrosis; glomerular hypertension, Renal
(anuric) sclerosis; tubular dilatation NIDDM, Failure with protein
casts and heart disease HK019 52 yr/F secondary to (D) CV/ 127 5.7
14.5 (CC) 23.7 8.4 12.4 >300 diffuse moderate glomerular
hypertension, Renal obsolescence with thickening NIDDM, Failure of
Bowman's capsule; peri- and heart glomerular fibrosis; moderate
disease tubular injury with diffuse tubulo-interstitial fibrosis,
tubular dilatation with protein casts. HK020 54 yr/F auto-immune
(D) CV/ 94 16.6 4.35 (CC) 29 9.6 5.4 na Severe end-stage renal
glomerulone- Stroke (anuric) disease; no functional phritis
glorneruli observed; severe glomerular sclerosis and interstitial
fibrosis with chronic inflammation; tubular congestion with protein
casts. HK021 15 mo/M no renal (D) Trauma 11 0.4 73.4 (CC) 29 10.3
3.4 trace normal kidney histology disease HK022 60 yr/M secondary
to (D) CVA/ 53 3.3 17 31.1 10 1.8 100 Severe end-stage renal
hypertension, Intracranial (GFR) disease; diffuse severe NIDDM
hemorrhage glomerulosclerosis; and heart interstitial fibrosis and
tubular disease atrophy with protein casts HK023 18 yr/M focal (KR)
failed 28 6.4 13.8 (GFR) 36 11.8 6.4 na focal segmental segmental
kidneys glomerulosclerosis (10-15% glomerulo- removed of glomeruli
sclerosed), sclerosis, prior to associated with diffuse nephrotic
transplant mesangial hypercellularity; syndrome, diffuse, focally
accentuated hypertension moderate to marked interstitial fibrosis
and tubular atrophy; marked chronic active interstitial
nephritis
Comparative functional analysis of albumin transport between
tubular-enriched B2 and tubular cell-depleted B4 subtractions is
shown in FIG. 8. Subtraction B2 is enriched in proximal tubule
cells and thus exhibits increased albumin-transport function.
Albumin uptake: Culture media of cells grown to confluency in
24-well, collagen IV plates (BD Biocoat.TM.) was replaced for 18-24
hours with phenol red-free, serum-free, low-glucose DMEM (pr-/s-/Ig
DMEM) containing 1.times. antimycotic/antibiotic and 2 mM
glutamine. Immediately prior to assay, cells were washed and
incubated for 30 minutes with pr-/s-/Ig DMEM+10 mM HEPES, 2 mM
glutamine, 1.8 mM CaCl2, and 1 mM MgCl2. Cells were exposed to 25
.mu.g/mL rhodamine-conjugated bovine albumin (Invitrogen) for 30
min, washed with ice cold PBS to stop endocytosis and fixed
immediately with 2% paraformaldehyde containing 25 .mu.g/mL Hoechst
nuclear dye. For inhibition experiments, 1 .mu.M
receptor-associated protein (RAP) (Ray Biotech, Inc., Norcross Ga.)
was added 10 minutes prior to albumin addition. Microscopic imaging
and analysis was performed with a BD Pathway.TM. 855 High-Content
BioImager (Becton Dickinson) (see Kelley et al. Am J Physiol Renal
Physiol. 2010 November; 299(5):F1026-39. Epub Sep. 8, 2010).
In conclusion, HCA yields cellular level data and can reveal
populations dynamics that are undetectable by other assays, i.e.,
gene or protein expression. A quantifiable ex-vivo HCA assay for
measuring albumin transport (HCA-AT) function can be utilized to
characterize human renal tubular cells as components of human NKA
prototypes. HCA-AT enabled comparative evaluation of cellular
function, showing that albumin transport-competent cells were
retained in NKA cultures derived from human CKD kidneys. It was
also shown that specific subtractions of NKA cultures, B2 and B4,
were distinct in phenotype and function, with B2 representing a
tubular cell-enriched fraction with enhanced albumin transport
activity. The B2 cell subpopulation from human CKD are
phenotypically and functionally analogous to rodent B2 cells that
demonstrated efficacy in vivo (as shown above).
Example 11--Marker Expression as a Predictor of Renal
Regeneration
This study concerns stem and progenitor marker expression as a
predictor of renal regeneration in 5/6 nephrectomized rats treated
with therapeutically bio-active primary renal cell sub-populations.
The underlying mechanisms by which NKA treatment improved renal
function are being characterized. Our studies on NKA treatment's
mechanism of action concern cell-cell signalling, engraftment, and
fibrotic pathways. The present work focused on how NKA treatment
might increase the organ's intrinsic regenerative capacity perhaps
by mobilizing renal stem cells. We hypothesize that the extended
survival and improvement in renal function observed in NKA-treated
5/6 NX rats is associated with molecular expression of specific
stem cell markers.
Using a rat 5/6 nephrectomy model for CKD, this study employs
molecular assays to evaluate the mobilization of resident stem and
progenitor cells within the rat 5/6 nephrectomized kidney in
response to direct injection with defined, therapeutically
bio-active primary renal cell populations. It was observed that
this cell-based therapy is specifically associated with
up-regulation of the key stem cell markers CD24, CD133, UTF1, SOX2,
LEFTY1, and NODAL at both transcript and protein levels.
Up-regulation was detected by 1 week post-injection and peaked by
12 weeks post-injection. Activation of stem and progenitor cell
markers was associated with increased survival and significant
improvement of serum biomarkers relative to untreated
nephrectomized controls.
Materials and Methods
Isolation of primary renal cell populations from rat. Isolation of
primary renal cell populations from rat were performed as
previously described (Aboushwareb et al., supra 2008; Presnell et
al., 2009 FASEB J 23: LB143).
In vivo study design and analysis. Detailed descriptions of the
isolation of primary renal cell populations (Presnell et al. Tissue
Eng Part C Methods. 2010 Oct. 27. [Epub ahead of print]) and the in
vivo studies that evaluated the bioactivity of primary renal cell
sub-populations in the 5/6 nephrectomized rodent model of CKD
(Kelley et al. supra 2010). A tubular cell-enriched subpopulation
of primary renal cells improves survival and augments kidney
function in a rodent model of chronic kidney disease were published
elsewhere. In the current study, tissues were isolated at necropsy
from rats treated with B2 (NKA #1) or a B2+B4 mixture (NKA #42) and
compared to nephrectomized (Nx) and sham-operated,
non-nephrectomized rats (Control). In FIGS. 9 and 11 and Table
11.1, data from NKA #1 and NKA #2 treated rats was pooled. Systemic
data was obtained by analysis of blood samples drawn weekly and
pre-necropsy from rats on study.
Table 11.1 shows survival data for sham treated animals (control),
n=3; nx control (Nx), n+3, animals treated with B2 cells (NKA #1),
n=7; B2+B4 cells (NKA #2); n=7. At the end of the study (23-24
weeks) none of the Nx animals remained. NKA treated animals had a
superior survival rate compared to the untreated Nx control.
TABLE-US-00008 TABLE 11.1 Early Midpoint End of Study Treatment
Group 1 week 12-13 Week [23-24 Week] Control 2/3* 2/2 2/2 NX 2/3*
1/2 0/1 NKA #1 5/7** 3/7** 1/3** NKA #2 5/7** 3/7** 3/3 *1 animal
sacrificed at scheduled timepoint for tissue **2 animals sacrificed
at scheduled timepoint for tissue
RNA Isolation, cDNA Synthesis and qRT-PCR. RNA was isolated from
tissues embedded in optimum cutting temperature (OCT) freezing
media as follows: tissue blocks were placed at room temperature and
excess OCT was removed, the tissues were then placed in PBS to
allow complete thawing and removal of residual OCT, the tissues
were washed three times in PBS and then coarsely chopped and
aliquoted into microfuge tubes. The aliquoted tissues were then
pulverized using a pestle and RNA was extracted using the RNeasy
Plus Mini Kit (Qiagen, Valencia Calif.). RNA integrity was
determined spectrophotomerically and cDNA was generated from a
volume of RNA equal to 1.4 .mu.g using the SuperScript.RTM.
VILO.TM. cDNA Synthesis Kit (Invitrogen, Carlsbad Calif.).
Following cDNA synthesis, each sample was diluted 1:6 by adding 200
.mu.l of diH.sub.2O to bring the final volume to 240 .mu.l. The
expression levels of target transcripts were examined via
quantitative real-time PCR (qRT-PCR) using catalogued primers and
probes from ABI and an ABI-Prism 7300 Real Time PCR, System
(Applied Biosystems, Foster City Calif.). Amplification was
performed using the TaqMan.RTM. Gene Expression Master Mix (ABI,
Cat #4369016) and peptidylprolyl isomerase B (PPIB) was utilized as
the endogenous control. qRT-PCR Reaction: 10 .mu.l Master Mix
(2.times.), 1 .mu.l Primer and Probe (20.times.), 9 .mu.l cDNA, 20
.mu.l Total Volume per Reaction. Each reaction was setup as follows
using the TaqMan.RTM. primers and probes.
TABLE-US-00009 Gene Abbreviation TaqMan primer SRY (sex determining
Sox2 Rn01286286_g1 region Y)-box 2 Undifferentiated Embryonic Cell
UTF1 Rn01498190_g1 Transcription Factor Nodal Homolog from Mouse
NODAL Rn01433623_m1 Prominin 1 CD133 Rn00572720_m1 CD24 CD24
Rn00562598_m1 LEFTY1
Western Blot. Frozen whole kidney tissue embedded in OCT freezing
media was utilized for protein sample collection. OCT was removed
as described above and all tissues were lysed in a buffer
consisting of 50 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP40, and
protease inhibitor cocktail (Roche Applied Science, Indianapolis
Ind.). Lysis proceeded for 15 minutes at room temperature with
rocking followed by centrifugation for 10 minutes at 13,000 RPM.
All supernatants were collected and protein concentrations were
determined by Bradford Assay. SDS PAGE Gel was carried out by
adding 30 .mu.g of protein per sample to each well of NuPAGE.RTM.
Novex 10% Bis-Tris Gels (Invitrogen). The gels were electrophoresed
for 40 min at 200V in MES running buffer (Invitrogen). The proteins
were then transferred to nitrocellulose membranes using the I-Blot
system (Invitrogen), and blocked with 15 mL of 4% w/v low-fat milk
dissolved in Tris Buffered Saline with 0.1% Tween-20 (TBS-T)
(Sigma, St. Louis, Mo.) for 2 hours at room temperature. The
membranes were probed overnight at room temperature with the
following antibodies: each diluted in 5 mL TBS-T with 2% w/v
low-fat milk. (Anti-Human Lefty-A Long & Short isoforms
(R&D systems MAB7461); Anti-Human, Mouse & Rat CD133 (Abcam
AB19898); Anti-Human & Mouse UTF1 (Millipore MAB4337);
Anti-Human NODAL (Abcam AB55676); Anti-Human & Rat CDH11 (OB
Cadherin) (Thermo Scientific MAI-06306); Anti-Rat CD24 (Becton
Dickinson)). The membranes were washed 3 times/10 minutes each with
TBS-T, then probed with the appropriate HRP-conjugated secondary
antibody (Vector Labs PI-2000; PI-1000) diluted in TBS-T with 2%
w/v low-fat milk (1:60,000) for 1.5 hours at room temperature. The
membranes were washed 3 times/10 minutes each in TBS-T, followed by
two 10-minute washes in diH.sub.2O. The blots were developed using
ECL Advance chemiluminescent reagent (GE Healthcare Life Sciences,
Piscataway N.J.) and visualized using the ChemiDoc.TM. XRS
molecular imager and Quantity One.RTM. software (BioRad, Hercules
Calif.).
Results. Molecular assays to evaluate the mobilization of resident
stem and progenitor cells in 5/6 NX rats were developed and used to
investigate the temporal response of these markers to NKA
treatment. It was observed that NKA treatment was specifically
associated with up-regulation of the key stem cell markers CD24,
CD133, UTF-1, SOX-2, LEFTY, and NODAL at mRNA transcript and
protein levels. Up-regulation was detected by 1 week post-injection
and had peaked by 12 weeks post-injection. Activation of stem and
progenitor cell markers was associated with increased survival and
significant improvement of serum biomarkers (i.e., improvement of
renal filtration) relative to untreated 5/6 NX control animals.
FIG. 9 shows the expression of SOX2 mRNA in host tissue after
treatment of 5/6 NX rats with NKA. Temporal analysis of SOX2 mRNA
expression showed 1.8-fold increase in SOX2 mRNA within NKA
treatment group over Nx control by 12 week post-implantation. A
2.7-fold increase in SOX2 mRNA expression was observed in NKA
treatment group over Nx control by 24 weeks post-implantation.
(1-week n=3 each for Control (sham), Nx (control), and NKA treated)
(12 week n=1 each for Control (sham) and Nx (control); NKA treated
n=4) (24 week n=1 each for Control (sham) and Nx (control); NKA
treated n=4). *Indicates p-value=0.023 or <0.05.
FIG. 10--Western blot showing time course of expression of CD24,
CD133, UTF1, SOX2, NODAL and LEFTY in sham control (Control). Nx
control (Nx), and rats treated NKA #1 and NKA #2 at 1, 12 and 24
weeks post-treatment. Frozen whole kidney tissue (N=1 for each
sample) embedded in OCT freezing media was utilized for protein
sample collection. Lanes were normalized by total mass protein
loaded. CD133, UTF1, NODAL, LEFTY and SOX2 protein levels in
NKA-treated tissues were elevated relative to Control or Nx rats at
all time points.
FIG. 11 depicts a time course of regenerative response index (RRI).
A densitometric analysis of individual protein expression (FIG. 10)
was used to generate a quantitative index of regenerative marker
protein expression, or regenerative response index (RRI). Band
intensity was calculated from each western blot using Image J v1.4
software (NIH) and values normalized per unit area for each
protein. Average intensity was determined for sham, Nx, and NKA
treatment groups by compiling the 5 markers used in the western
blot analysis for each time point. Plot shows XY scatter with
smoothed line fit generated from 1, 12, and 24 week time points.
The average intensity for each group was plotted over time to
highlight the trends in the host tissue response of stem cell
marker protein expression. Statistical analysis was performed using
standard two tailed Student's t-test assuming equal variance for
each sample. Confidence interval of 95% (p-value<0.05) was used
to determine statistical significance. (NKA treated group n=2;
Control (sham) n=1; Nx (control) n=1). In sham control animals, RRI
shows only a slight reduction from 90.47 at 1 week post-treatment
to 81.89 at 24 weeks post treatment. In contrast, kidney from 5/6
Nx controls presents essentially the opposite response, with RRI
increasing from 82.26 at 1 week post-treatment to 140.56 at 18
weeks post-treatment, at which point the animal died. In
NKA-treated animals RRI increased sharply from 62.89 at 1 week
post-treatment to 135.61 by 12 weeks post-treatment and fell to
112.61 by 24 weeks post-treatment.
NKA treatment was observed to be associated with up-regulation of
the stem cell markers CD24, CD133, UTF-1, SOX-2 and NODAL at both
transcript and protein levels in the host tissue. Up-regulation was
detected by 1 week post-treatment and peaked by 12 weeks
post-treatment. Overall activation of stem and progenitor cell
markers in host tissues was associated with increased survival (1)
and improvement of clinically-relevant serum biomarkers relative to
untreated nephrectomized controls.
Mobilization of resident stem, and progenitor cell populations in
response to NKA treatment may contribute to the restoration of
kidney function in 5/6 NX animals by regenerating damaged kidney
tissue and organ architecture. The molecular assays used in this
study might therefore provide a rapid, straightforward, and
predictive assay of regenerative outcomes for evaluating tissue
engineering and regenerative medicine treatments for CKD.
Example 12--Exosomes Derived from Primary Renal Cells Contain
MicroRNAs
We sought to correlate specific exosome-derived miRNAs with
functionally-relevant outcomes in target cells in vitro to inform
the design of in vivo studies for elucidating mechanisms that yield
regenerative outcomes.
METHODS: The effect of conditioned media on signaling pathways
associated with regenerative healing responses was investigated
using commercially available cells: HK-2 (human proximal tubule
cell line), primary human renal mesangial cells (HRMC), and human
umbilical cord endothelial cells (HUVEC). RNA content from exosomes
in conditioned media from human and rat primary renal cell cultures
(UNFX) was screened by PCR-based array designed to detect known
miRNAs. Low oxygen has been reported to affect exosome shedding;
therefore, a group of cultures was exposed to low oxygen (2%
O.sub.2) for 24 hours prior to media collection. Exosomes were
separated from cellular debris by FACS.
FIG. 12 provides a schematic for the preparation and analysis of
UNFX conditioned media.
RESULTS: UNFX-conditioned media was found to affect signaling
pathways associated with regenerative healing responses; these
responses were not observed in controls using non-conditioned
media. Specifically, NF.kappa.B (immune response) and
epithelial-to-mesenchymal transition (fibrotic response) was
attenuated in HK-2 cells, PAI-1 (fibrotic response) was attenuated
in HRMC cells, and angiogenesis was promoted in HUVEC. Preliminary
data from PCR array screening of exosome content from
UNFX-conditioned media indicates that UNFX produces exosomes
containing miRNA sequences consistent with the observed responses
to UNFX-conditioned media.
FIGS. 13A-C show that conditioned media from UNFX cultures affects
multiple cellular processes in vitro that are potentially
associated with regenerative outcomes. NFkB signaling is proposed
as a key mediator of inflammatory processes in kidney diseases
(Rangan et al., 2009. Front Biosci 12:3496-3522; Sanz et al., 2010.
J Am Soc Nephrol 21:1254-1262), and can be activated by Tumor
Necrosis Factors (TNF). HK-2 cells were preincubated with
unconditioned media (left) or UNFX conditioned media (right) for 1
hour at 37.degree. C., then activated with or without 10 ng/ml
TNFa.
FIG. 13A shows that UNFX-conditioned media attenuates TNF-a
mediated activation of NF-kB. NFkB activation was measured by
RelA/p65 immunofluorescence staining (green).
Hoechst-counter-stained nuclei (blue) and phalloidin-stained
filamentous actin (red) facilitate assessment of RelA/p65 nuclear
localization (white arrows).
FIG. 13B shows that UNFX-conditioned media increases proangiogenic
behavior of HUVEC cell cultures. HUVEC cells (100,000 per well)
were overlaid onto polymerized Matrigel in Media 200 plus 0.5% BSA.
Unconditioned media (left) or UNFX-conditioned medium (right) was
added and cellular organizational response was monitored visually
for 3-6 hours with image capture. Cellular organization was scored
for cell migration (white arrowheads), alignment (black
arrowheads), tubule formation (red arrowheads), and formation of
closed polygons (asterisks). UNFX conditioned media induced more
tubules and closed polygons compared to unconditioned media,
suggesting that proangiogenic factors are present in the media.
FIG. 13C shows that UNFX-conditioned media attenuates fibrosis
pathways in epithelial cells. HK-2 cells lose epithelial
characteristics, and acquire a mesenchymal phenotype when exposed
to Transforming Growth Factors (TGF) in vitro, replicating the
epithelial-to-mesenchymal transition (EMT) that is associated with
progression of renal fibrosis (Zeisberg et al. 2003 Nat Med
9:964-968). HK-2 cells were cultured in unconditioned media (CTRL),
unconditioned media containing 10 ng/ml TGF.beta.1 (TGF.beta.1), or
UNFX conditioned media containing 10 ng/ml TGF.beta.1
(TGF.beta.1+CM) for 72 hours. Cells were assayed by quantitative
RT-PCR for CDH1 (epithelial marker), CNN1 (mesenchymal marker) and
MYH11 (mesenchymal marker). Conditioned media reduces the degree of
TGF.beta.1-induced EMT as measured by CDH1, CNN1, and MYH11 gene
expression. Error bars represent the standard error of the mean
(SEM) of three experimental replicates.
FIG. 13D depicts the positive feedback loop established by
TGF.beta.1 and Plasminogen Activator Inhibitor-1 (PAI-1) that, when
left unchecked, can lead to the progressive accumulation of
extracellular matrix proteins (Seo et al., 2009. Am J Nephrol
30:481-490).
FIGS. 14A-B show the attenuation of fibrosis pathways in mesangial
cells. HRMC were cultured for 24 hours in control (CTRL) or UNFX
conditioned media (LTNFX CM) with (+) or without (-) the addition
of 5 ng/ml TGF.beta.1. Western blot analysis for PAI-1 demonstrates
that UNFX CM attenuates the TGF.beta.1-induced increase in PAI-1
protein levels. bActin is shown as a loading control. Human renal
mesangial cells (HRMC) express increased levels of PAI-1 in the
presence (+) of 5 ng/ml TGFb1. Co-culture with conditioned media
(CM) derived from human bioactive kidney cells attenuates
TGFb1-induced PAI-1 protein expression. PAI-1 expression at the
mRNA level was unaltered by CM (data not shown).
FIG. 14B shows that CM from rat bioactive kidney cells had similar
effect on cultured HRMC induced (+) and uninduced (-) with TGFb1.
CM supernatant (Deplete Rat CM) collected after centrifugation was
less effective at attenuating PAI-1 expression, suggesting that the
CM component responsible for the observed attenuation of PAI-1
protein might be associated with vesicles secreted by the rat
bioactive kidney cells.
FIGS. 15A-C show that the conditioned media from UNFX contains
secreted vesicles. FIG. 15 depicts secreted vesicles (including
exosomes), which are bilipid structures (red) that encompass
cytoplasm-derived internal components (green). Phosphatidylserines
(blue triangles) are components of the membrane that are exposed to
the extracellular space during vesicle biogenesis (Thery et al.,
2010. Nat Rev Immunol 9:581-593).
PKH26 and CFSE label the lipid membrane and cytoplasm of secreted
vesicles (Aliotta et al., 2010. Exp Hematol 38:233-245),
respectively, while Annexin V binds phosphatidylserines.
FIGS. 15B-C shows FACS sorting. UNFX conditioned media was labeled
with PKH-126, CFSE, and APC-conjugated Annexin V, then sorted by
fluorescence-assisted cell sorting (FACS). Triple-positive
particles, representing secreted vesicles, were collected and total
RNA was extracted using TRIZol reagent, microRNA content was
screened for known sequences using commercially available
RT-PCR-based arrays.
Table 12.1 shows that secreted vesicles contain microRNAs with
predicted therapeutic outcomes. UNFX cells shed exosomes that
contain known miRNA sequences. UNFX-conditioned media affects
functionally-relevant regenerative responses in human cell lines.
The cause and effect relationship between detected miRNAs and
observed regenerative responses is under active investigation;
however, the results achieved to date suggest that UNFX cells have
the potential to produce therapeutically-relevant paracrine effects
via exosome-mediated transfer of miRNAs to target cells and
tissues.
TABLE-US-00010 TABLE 12.1 miRNA in exosomes Gene targets Predicted
effects miR-146a TRAF6, IRAK1* Inhibits NFkB miR-130a GAX, HOXA5**
Promotes angiogenesis miR-23b Smad 3/4/5*** Inhibits TGF.beta.
signal transduction (anti-fibrotic) *Taganov et al, 2006. Proc Natl
Acad Sci USA 103: 12481-12486. **Chen and Gorski, 2008. Blood 111:
1217-1226. ***Rogler et al., 2009. Hepatology 50: 575-584.
The data support the conclusion that excreted vesicles from
bioactive renal cell cultures contain components that attenuate
PAI-1 induced by the TGFb1/PAI-1 feedback loop.
Microarray and RT-PCR analysis. Unfractionated (UNFX) bioactive
renal cells from Lewis rats were cultured in basal media (50:50 mix
of DMEM and KSFM without serum or supplements) for 24 hours under
low oxygen conditions (2% O2). Conditioned media was collected and
ultracentrifuged at 100,000.times.g for 2 hours at 4 C to pellet
secreted vesicles (e.g. microvesicles, exosomes). Total RNA was
extracted from the resulting pellet, and assayed for known microRNA
species by real time RT-PCR (Rat MicroRNA Genome V2.0 PCR Array;
Qiagen #MAR-100A). The following miRNAs were detectable.
TABLE-US-00011 miR-21 miR-10b let-7f miR-23a miR-27b miR-181d
miR-30c miR-125a-5p miR-181a miR-1224 miR-30d miR-221 miR-23b
miR-31 miR-30a* miR-92a miR-93 miR-351 miR-100 miR-182 miR-218
miR-125b-5p miR-99a miR-210 miR-195 miR-320 miR-98 miR-10a-5p
miR-664 miR-18a miR-370 miR-30e* miR-342-3p miR-24 let-7i miR-203
miR-30a miR-196a miR-352 miR-16 miR-26b miR-181c miR-126* miR-200a
miR-222 miR-30b-5p miR-126 miR-219-1-3p miR-27a miR-29c miR-708
miR-20a miR-200c miR-652 let-7c miR-151 let-7d* miR-26a miR-429
miR-503 miR-17-5p miR-103 miR-138 miR-30e let-7a miR-450a miR-25
miR-322* miR-365 let-7b miR-15b miR-874 miR-20b-5p miR-378
miR-345-5p miR-29a miR-127 miR-374 let-7d miR-199a-5p miR-872
miR-22 miR-181b miR-186 miR-322 miR-106b miR-130a let-7e miR-196c
miR-140* miR-191 miR-196b miR-28* miR-99b miR-19a miR-212 miR-19b
miR-145 miR-139-3p miR-347 miR-96 miR-871 miR-151* miR-34a miR-29a*
miR-328 miR-223 miR-542-5p miR-185 miR-301b miR-129 miR-28 miR-505
miR-214 miR-192 miR-532-3p miR-29c* miR-92b miR-7a miR-489 miR-672
miR-451 miR-141 miR-150 miR-34c* miR-500 miR-425 miR-339-3p
miR-17-3p miR-146a miR-190 miR-339-5p miR-107 miR-671 miR-7b
miR-330* miR-465 miR-501 miR-409-3p miR-674-3p miR-206 miR-877
miR-21* miR-193* miR-760-3p miR-99b* miR-350 miR-770 miR-125a-3p
miR-466b miR-152 miR-183 miR-205 miR-106b* miR-143 miR-598-5p
miR-675 miR-324-5p miR-532-5p miR-423 miR-674-5p miR-411 miR-194
miR-760-5p miR-188 miR-490 miR-361 miR-331 miR-128 miR-296 miR-124
miR-497 miR-148b-3p miR-431 miR-301a miR-542-3p miR-154 miR-130b
miR-667 miR-30d* miR-199a-3p miR-935 miR-466c miR-326 miR-24-2*
miR-142-3p miR-132 miR-433 miR-7a* miR-375 miR-295 miR-298 miR-25*
miR-140 let-7b* miR-22* miR-9 miR-338 miR-449a miR-421 miR-384-5p
miR-125b* miR-485 miR-344-5p miR-34c miR-296* miR-133a miR-346
miR-29b miR-582 miR-147 miR-382 miR-541 miR-9* miR-511 miR-344-3p
miR-146b miR-99a* miR-345-3p miR-219-5p miR-499 miR-362 miR-653
miR-101a* miR-380 miR-340-3p miR-20b-3p miR-878 miR-224 miR-434
miR-377 miR-330 miR-139-5p miR-409-5p miR-544 miR-10a-3p miR-216a
miR-193 miR-148b-5p miR-496 miR-761 miR-598-3p miR-379 miR-181a*
miR-29b-2* miR-711 miR-27a* miR-335 miR-32 miR-34b miR-215
miR-291a-3p miR-455 miR-219-2-3p miR-487b miR-20a* miR-327 miR-23a*
miR-488 miR-204 miR-294 miR-26b* miR-504 miR-207 miR-342-5p let-7e*
miR-343 miR-543 miR-129* miR-292-3p miR-105 miR-101b miR-376a
miR-291a-5p miR-122 miR-540 miR-138* miR-539 miR-336 miR-190b
miR-30b-3p miR-134 let-7i* miR-184 miR-24-1* miR-484 miR-673
miR-363 miR-30c-2* miR-463 miR-329 miR-125b-3p miR-297 miR-133b
miR-764 miR-363* miR-875 miR-201 miR-153 miR-369-5p miR-483
miR-743a miR-376b-3p miR-742 miR-323 miR-211 miR-1 miR-495 miR-33
miR-293 miR-410 miR-758 miR-349 miR-137 miR-802 miR-30c-1*
miR-300-3p
Example 13--Paracrine Factors Derived from Bioactive Kidney
Cells
In the present study, we employed in vitro cell-based assays to
investigate potential paracrine mechanism(s) by which bioactive
kidney cells could modulate fibrosis through mediators such as
Plasminogen Activator Inhibitor-1 (PAI-1).
Materials and Methods: Conditioned media was collected from rat and
human cultures of bioactive kidney cells (Aboushwareb et al., World
J Urol 26, 295, 2008, Presnell et al. 2010 supra) under serum- and
supplement-free conditions and utilized for in vitro assays.
Commercially available rat- and human-derived mesangial cells were
used as surrogates for host-response tissues in the in vitro assays
because mesangial cells are a source of PAI-1 production in injured
or diseased kidneys (Rerolle et al., Kidney Int 58, 1841, 2000.).
PAI-1 gene and protein expression were assayed by quantitative
RT-PCR and Western blot, respectively. Vesicular particles shed by
cells into the culture media (e.g., exosomes) were collected by
high-speed centrifugation (Wang et al., Nuc Acids Res 2010, 1-12
doi:10.1093/nar/gkq601, Jul. 7, 2010) and total RNA extracted from
the pellet with TRIzol reagent (Invitrogen). RNA content of the
vesicles was screened using PCR-based arrays of known microRNA
sequences (Qiagen).
Results: Conditioned media from bioactive kidney cell cultures
attenuated the TGF.beta.1-induced increase in PAI-1 steady-state
protein levels in mesangial cells, but did not affect steady state
mRNA levels; an observation that is consistent with the mechanism
by which microRNAs modulate target genes. Based on the hypothesis
that microRNAs can be transferred between cells through
extracellular vesicle trafficking (Wang et al., supra 2010), we
analyzed the conditioned media for microRNA content and confirmed
the presence of microRNA 30b-5p (miR-30b-5p), a putative inhibitor
of PAI-1.
The data presented here suggest that bioactive kidney cells may
modulate fibrosis directly through cell-to-cell transfer of
miR-30b-5p to target mesangial cells via exosomes. As a result of
miR-30b-5p uptake by mesangial cells, TGF.beta.1-induced increases
in steady-state PAI-1 protein levels are attenuated, a response
that, in renal tissue, could ultimately reduce deposition of
extracellular matrix within the glomerular space. Current work is
underway to confirm that PAI-1 is indeed a direct target of
miR-30b-5p.
FIGS. 14A-B show a western blot of PAI-1 and .alpha.-Actin
(control) protein expression in human mesangial cells cultured for
24 hour in control (CTRL) or bioactive kidney cell conditioned
media (CM) with (+) or without (-) TGF.beta.1 addition to the
culture media. In CTRL cultures, TGF.beta.1 increased PAI-1 protein
expression. In CM cultures, the TGF.beta.1-induced response was
attenuated.
Secreted vesicles were analyzed for microRNAs that may be putative
repressors of PAI-1. Secreted vesicles from human and rat bioactive
kidney cell CM were collected by high-speed centrifugation and
assayed for microRNA content using PCR-based arrays of known
sequences. miR-449a, a putative regulator of PAI-1 (6), was
identified. HMRC were transiently transfected with miR-449a or not
(CTRL). 24 hours post-transfection cells were either exposed to 5
ng/ml TGFb1 (+) or not (-) for an additional 24 hours.
FIG. 16A shows a Western blot in which total protein was prepared
and assayed for PAI-1 and bActin. miR-449a reduced steady-state
PAI-1 protein levels (compare lane 1 to lane 3) and induced levels
of PAI-1 protein were also lower in miR-449a transfected cultures
(compare lane 2 to lane 4). The data support the conclusion that
excreted vesicles contain miR-449a and uptake of miR-449a into
mesangial cells reduces PAI-1 expression.
FIG. 16B depicts the microRNA, miR-30b-5p, which was also
identified in the PCR-based array and is a putative regulator of
PAI-1 based on predictive algorithms (http://mirbase.org--miRBase
is hosted and maintained in the Faculty of Life Sciences at the
University of Manchester).
PAI-1 protein levels in glomeruli were examined in vivo after
treatment of CKD induced by 5/6 nephrectomy with bioactive renal
cells.
FIGS. 17A-C show representative immunohistochemistry images of
PAI-1 (A-C) in Lewis rat kidneys that have undergone unilateral
nephrectomy (A), 5/6 nephrectomy (B), or 5/6 nephrectomy with
intra-renal delivery of bioactive kidney cells (C). Accumulation of
PAI-1 in the glomerulus (arrowheads) as a result of the 5/6
nephrectomy procedure (B) was reduced as a result of treatment
(C).
In a separate study, qRT-PCR was conducted on kidney tissue
harvested at necropsy and the relative gene expression values were
plotted against days on study.
FIG. 17D shows that 5/6 nephrectomized rats (red squares)
demonstrated more robust expression of PAI-1 relative to those
treated with bioactive renal cells (blue diamonds) and
sham-operated controls (green triangles).
FIG. 17E shows representative Western blot analysis on kidney
samples taken at 3 and 6 months post-treatment. Treated tissues
(Nx+Tx) of 5/6 nephrectomized rats (Nx) had reduced the
accumulation of PAI-1 and Fibronectin (FN) protein (Kelley et al.
2010 supra).
The data support the conclusion that in vivo PAI-1 protein levels
in glomeruli decrease after treatment of CKD induced by 5/6
nephrectomy with bioactive renal cells.
When taken together, Examples 12-13 support the hypothesis that one
mechanism by which intra-renal delivery of bioactive kidney cells
improves renal function might be via cell-cell transfer of
components that modulate fibrotic pathways in resident kidney
cells.
Example 14--Secreted Factors from Bioactive Kidney Cells Attenuate
NF.kappa.B Signaling Pathways
In this study, we investigated the role of NF.kappa.B pathways in
the NKA-mediated attenuation of disease progression in the 5/6
nephrectomy model and to identify properties of the bioactive
kidney cells that may contribute to regenerative outcomes through
direct modulation of NF.kappa.B activation. FIG. 17G depicts the
canonical activation of the NFkB pathway by TNF.alpha..
Materials and Methods: Remnant kidneys were harvested from Lewis
rats in which a two-step 5/6 nephrectomy procedure was performed 6
weeks prior to being treated with B2+B4 in PBS (NKA prototype).
NKA-treated (TX) or untreated (UNTX) tissues were assayed for
NF.kappa.B activation by immunohistochemistry, RT-PCR, Western blot
analysis, and electrophoresis mobility shift assays (EMSA).
Conditioned media (CM) collected from ex vivo NKA cell cultures
grown in serum- and supplement-free media was used for in vitro
functional assays. The human proximal tubule cell line (HK-2) was
used as target cell type for molecular and immunofluorsence-based
assay readouts. Vesicular particles shed by cells into the culture
media (exosomes) were collected by high-speed centrifugation. Total
RNA isolated from exosomes was screened using PCR-based arrays of
known microRNA sequences (Qiagen).
Results: Nuclear localization of the NF.kappa.B subunit, RelA/p65,
was observed in remnant kidneys from 5/6 nephrectomized rats,
suggesting activation of inflammatory pathways in UNTX tissues.
Preliminary comparison with TX tissues by RT-PCR showed a decrease
in RelA gene expression, suggesting that NKA treatment may
influence NF.kappa.B pathway activation through inhibition of
RelA/p65 expression. This hypothesis is supported by the
observation that CM attenuates TNF.alpha.-induced NF.kappa.B
activation in vitro, as evidenced by the reduced nuclear
localization of RelA/p65 in CM-exposed HK-2 cells (FIG. 17F)
relative to that seen in response to Tumor Necrosis Factor-.alpha.
(TNF .alpha.). Ongoing RT-PCR analyses of NKA exosome microRNAs are
investigating whether sequences known to influence NF.kappa.B
pathways are present.
FIG. 17F shows a 2-hour exposure to NKA CM reduces nuclear
localization of NF.kappa.B p65 (green) in HK-2 compared to that
observed in control cultures pretreated with TNF.alpha. in
immunofluorescent assays. In HK-2, NFkB p65 (green) localizes to
the nucleus after a 30 minute exposure to TNF.alpha. (Control
Media). However, pre-treatment of HK-2 cells with NKA Conditioned
Media for 2 hours prior to TNF.alpha. addition attenuated the NFkB
p65 nuclear localization response. Nuclei are stained with DAPI
(blue) and filamentous actin is stained with Alexa594-phalloidin
(red) to assist in qualitatively assessing the robustness of
NF.kappa.B nuclear localization (note the slightly diminished
phalloidin borders in TNF.alpha.-treated control cells in the
merged panels in the bottom row). The counterstaing provide
reference for the NFkB localization in the merged images.
Immunohistochemistry for the NFkB p65 subunit in kidney tissues of
Lewis rats reveals that animals with progressive CKD initiated by
5/6 nephrectomy (panel B) have more robust nuclear localization of
NFkB p65 subunit, particularly in tubular epithelial cells (black
arrowheads)) relative to the non-progressive renal insufficiency
initiated by unilateral nephrectomy in control animals (panel A).
Tissues harvested six weeks post-nephrectomy. Magnification at
200.times..
Panel C: Western blot analysis for NFkB p65 in the cytoplasmic
(`C`) and nuclear (`N`) protein extracts of Lewis rat kidney tissue
that have undergone the 5/6 nephrectomy. Comparing weeks 1 and 13,
where gtubulin levels (loading control) are relatively consistent,
nuclear NFkB p65 increases over time, consistent with the
immunohistochemistry results.
Panel D: Electrophoretic mobility shift assay (EMSA) on nuclear
extracts confirms that the NFkB that localizes to the nucleus
following 5/6 nephrectomy is activated for DNA binding. Lanes
represent nuclear extracts prepared from two animals at each time
point.
The NFkB pathway is progressively activated in the 5/6 nephrectomy
model of chronic kidney disease. Immunohistochemistry for the NFkB
p65 subunit in kidney tissues of Lewis rats was performed.
FIGS. 18A-D reveal that animals with progressive CKD initiated by
5/6 nephrectomy (panel B) have more robust nuclear localization of
NFkB p65 subunit, particularly in tubular epithelial cells (black
arrowheads) relative to the non-progressive renal insufficiency
initiated by unilateral nephrectomy in control animals (panel A).
Tissues harvested six weeks post-nephrectomy. Magnification at
200.times..
FIG. 18C shows Western blot analysis for NFkB p65 in the
cytoplasmic (`C`) and nuclear (`N`) protein extracts of Lewis rat
kidney tissue that have undergone the 5/6 nephrectomy. Comparing
weeks 1 and 13, where gtubulin levels (loading control) are
relatively consistent, nuclear NFkB p65 increases over time,
consistent with the immunohistochemistry results.
FIG. 18D shows an electrophoretic mobility shift assay (EMSA) on
nuclear extracts and confirms that the NFkB that localizes, to the
nucleus following 5/6 nephrectomy is activated for DNA, binding.
Lanes represent nuclear extracts prepared from two animals at each
time point. 1 mg of nuclear protein was incubated with 5 ng of NFkB
DNA binding site, electrophoresed on a 6% DNA retardation gel, then
subsequently stained with ethidium bromide.
Intra-renal delivery of NKA cells reduces NFkB nuclear
localization. Multiple defined subpopulations of renal cells have
been isolated and assayed in vivo for bioactivity in improving
renal function in the 5/6 nephrectomy model of CKD (Presnell et al.
2010 supra). NKA cells demonstrated bioactivity whereas other
subpopulations did not (Kelley et al. 2010 supra).
FIG. 18E shows that Lewis rats with established CKD that received
intra-renal injection of NKA (A) or non-bioactive renal cells (B).
Lewis rats with established CKD received intra-renal injection of
NKA (A) or non-bioactive renal cells (B). At 6 months
post-treatment, tissues were harvested and assayed by
immunohistochemistry for the NFkB p65 subunit. Tissues from
NKA-treated animals exhibited less nuclear localization of NFkB
p65, particularly in the proximal tubules, compared to tissues from
animals treated with non-bioactive renal cells, suggesting that the
NKA treatment participated in attenuating the NFkB pathway activity
in vivo.
Analysis of microRNA content of secreted vesicles isolated from
human and rat NKA conditioned media by high-speed centrifugation
using PCR-based arrays of known sequences identified several
microRNA species that may influence immune responses via NFkB based
on literature reports (Marquez R T et al. (2010) Am J Physiol
Gastrointest Liver Physiol 298:G535; Taganov K D et al. (2006) Proc
Natl Acad Sci USA 103:12481) or predictive algorithms
(http://mirbase.org--miRBase is hosted and maintained in the
Faculty of Life Sciences at the University of Manchester).
TABLE-US-00012 microRNA in vesicles Target mRNA miR-21 Pellino-1
(Marquez et al.) miR-146a IRAK1, TRAF6 (Taganov et al.) miR-124,
miR-151 NFKB/RelA (miRBase)
The in vivo and in vitro findings provide insight on how bioactive
kidney cells (NKA) might improve renal function in
chronically-diseased kidneys by modulating immune response pathways
such as those affected by NFkB activation. Activated NFkB (p65
nuclear localization, particularly in proximal tubule cells) is
associated with the establishment of chronic kidney disease in the
5/6 nephrectomy rodent model and was attenuated by NKA treatment.
The in vitro response of proximal tubule cells (HK-2) to NKA
conditioned medium mimics the in vivo attenuation of NFkB nuclear
localization in response to NKA treatment. Putative mediators of
cell-cell inhibition of NFkB activation (microRNAs) were identified
in NKA conditioned medium. Taken together, these data support the
hypothesis that one mechanism by which intra-renal delivery of
bioactive kidney cells improves renal function might be via
cell-cell transfer of components, e.g., RNA, that modulate immune
responses in resident kidney cells.
Example 15--Functional Evaluation of NKA Constructs
Renal cell populations seeded onto gelatin or HA-based hydrogels
were viable and maintained a tubular epithelial functional
phenotype during an in vitro maturation of 3 days as measured by
transcriptomic, proteomic, secretomic and confocal
immunofluorescence assays. To investigate a potential mechanism by
which NKA Constructs could impact a disease state, the effect of
conditioned media on TGF-.beta. signaling pathways related to
tubulo-interstitial fibrosis associated with CKD progression was
evaluated. Conditioned medium was observed to attenuate
TGF-.beta.-induced epithelial-mesenchymal transition (EMT) in vitro
in a human proximal tubular cell line (HK2).
Materials and Methods.
Biomaterials. Biomaterials were prepared as beads (homogenous,
spherical configuration) or as particles (heterogenous population
with jagged edges). Gelatin beads (Cultispher S and Cultispher GL)
manufactured by Percell Biolytica (Astorp, Sweden) were purchased
from Sigma-Aldrich (St. Louis, Mo.) and Fisher Scientific
(Pittsburgh, Pa.), respectively. Crosslinked HA and HA/gelatin
(HyStem.TM. and Extracel.TM. from Glycosan BioSystems, Salt Lake
City, Utah) particles were formed from lyophilized sponges made
according to the manufacturer's instructions. Gelatin (Sigma)
particles were formed from crosslinked, lyophilized sponges.
PCL was purchased from Sigma-Aldrich (St. Louis, Mo.). PLGA 50:50
was purchased from Durect Corp. (Pelham, Ala.). PCL and PLGA beads
were prepared using a modified double emulsion (W/O/W) solvent
extraction method. PLGA particles were prepared using a solvent
casting porogen leaching technique. All beads and particles were
between 65 and 355 microns when measured in a dry state.
Cell isolation, preparation and culture. Cadaveric human kidneys
were procured through National Disease Research Institute (NDRI) in
compliance with all NIH guidelines governing the use of human
tissues for research purposes. Canine kidneys were procured from a
contract research organization (Integra). Rat kidneys (21 day old
Lewis) were obtained from Charles River Labs (MI). The preparation
of primary renal cell populations (UNFX) and defined
sub-populations (B2) from whole rat, canine and human kidney has
been previously described (Aboushwareb et al. World J Urol
26(4):295-300; 2008; Kelley et al. supra 2010; Presnell et al.
WO/2010/056328). In brief, kidney tissue was dissociated
enzymatically in a buffer containing 4.0 units/mL, dispase (Stem
Cell Technologies, Inc., Vancouver BC, Canada) and 300 units/ml
collagenase IV (Worthington Biochemical, Lakewood N.J.), then red
blood cells and debris were removed by centrifugation through 15%
iodixanol (Optiprep.RTM., Axis Shield, Norton, Mass.) to yield
UNFX. UNFX cells were seeded onto tissue culture treated
polystyrene plates (NUNC, Rochester N.Y.) and cultured in 50:50
media, a 1:1 mixture of high glucose DMEM:Keratinocyte Serum Free
Medium (KSFM) containing 5% FBS, 2.5 .mu.g EGF, 25 mg BPE,
1.times.ITS (insulin/transferrin/sodium selenite medium
supplement), and antibiotic/antimycotic (all from Invitrogen,
Carlsbad Calif.). B2 cells were isolated from UNFX cultures by
centrifugation through a four-step iodixanol (OptiPrep; 60% w/v in
unsupplemented KSFM) density gradient layered specifically for
rodent (16%, 13%, 11%, and 7%), canine (16%, 11%, 10%, and 7%), or
human (16%, 11%, 9%. and 7%) (Presnell et al. WO/2010/056328;
Kellen et al. supra 2010). Gradients were centrifuged at
800.times.g for 20 minutes at room temperature (without brake).
Bands of interest were removed via pipette and washed twice in
sterile phosphate buffered saline (PBS).
Cell/biomaterial composites (NKA Constructs). For in vitro analysis
of cell functionality on biomaterials, a uniform layer of
biomaterials (prepared as described above) was layered onto one
well of a 6-well low attachment plate (Costar #3471, Corning).
Human UNFX or B2 cells (2.5.times.10.sup.5 per well) were seeded
directly onto the biomaterial. For studies of adherence of canine
cells to biomaterials, 2.5.times.10.sup.6 UNFX cells were seeded
with 50 .mu.l packed volume of biomaterials in a non-adherent
24-well plate (Costar #3473, Corning). After 4 hours on a rocking
platform, canine NKA Constructs were matured overnight at
37.degree. C. in a 5% CO.sub.2 incubator. The next day, live/dead
staining was performed using a live/dead staining assay kit
(Invitrogen) according to the manufacturer's instructions. Rat NKA
Constructs were prepared in a 60 cc syringe on a roller bottle
apparatus with a rotational speed of 1 RPM.
For the transcriptomic, secretomic, and proteomic analyses
described below, NKA Constructs were matured for 3 days. Cells were
then harvested for transcriptomic or proteomic analyses and
conditioned media was collected for secretomic profiling.
Functional analysis of tubular cell associated enzyme activity.
Canine NKA Constructs (10 .mu.l loose packed volume) in 24-well
plates were evaluated using an assay for leucine aminopeptidase
(LAP) activity adapted from a previously published method (Tate et
al. Methods Enzymol 113:400-419; 1985). Briefly, 0.5 ml of 0.3 mM
L-leucine p-nitroanalide (Sigma) in PBS was added to NKA Constructs
for 1 hour at room temperature. Wells were sampled in duplicate and
absorbance at 405 nm recorded as a measure of LAP activity. LLC-PK1
cell lysate (American Type Culture Collection, or ATCC) served as
the positive control.
Transcriptomic profiling. Poly-adenylated RNA was extracted using
the RNeasy Plus Mini Kit (Qiagen, CA). Concentration and integrity
was determined by UV spectrophotometry. cDNA was generated from 1.4
.mu.g isolated RNA using the SuperScript VILO cDNA Synthesis Kit
(Invitrogen). Expression levels of target transcripts were examined
by quantitative real-time polymerase chain reaction (qRT-PCR) using
commercially available primers and probes (Table 15.1) and an
ABI-Prism 7300 Real Time PCR System (Applied Biosystems, CA).
Amplification was performed using TaqMan Gene Expression Master Mix
(ABI, Cat #4369016) and TATA Box Binding Protein gene (TBP) served
as the endogenous control. Each reaction consisted of 10 .mu.l
Master Mix (2.times.), 1 .mu.l Primer and Probe (20.times.) and 9
.mu.l cDNA. Samples were run in triplicate.
TABLE-US-00013 TABLE 15.1 Human TaqMan Primers/Probes Gene Abbrv.
Marker TaqMan Cat # Aquaporin 2 AQP2 Distal Collecting Duct Tubule
Hs00166640_m1 Epithelial Cadherin/Cadherin 1, Type 1 CDH1/ECAD
Distal Tubule Hs00170423_m1 Neuronal Cadherin/Cadherin 2, Type 1
CDH2/NCAD Proximal Tubule Hs00169953_m1 Cubilin, Intrinsic
Factor-Cobalamin Receptor CUBN Proximal Tubule Hs00153607_m1
Nephrin NPHS1 Glomerular/Podocyte Hs00190466_m1 Podocin NPHS2
Glomerular/Podocyte Hs00922492_m1 Erthropoietin EPO Kidney
Interstitum Hs01071097_m1 Cytochrome P450, Family 24, CYP2R1
Proximal Tubule Hs01379776_m1 Subfamily A, Polypeptide 1/ Vitamin D
24-Hydroxylase Vascular Endothelial Growth Factor A VEGFA
Endothelial/Vascular Hs00900055_m1 Platelet/Endothelial Cell
Adhesion Molecule PECAM1 Endothelial/Vascular Hs00169777_m1 Smooth
Muscle Myosin Heavy Chain MYH11/SMMHC Smooth Muscle Hs00224610_m1
Calponin CNN1 Smooth Muscle Hs00154543_m1 TATA Box Binding Protein
TBP Endogenous Control Hs99999910_m1
Secretomic profiling. Conditioned medium from human NKA Constructs
was collected and frozen at -80.degree. C. Samples were evaluated
for biomarker concentration quantitation. The results for a given
biomarker concentration in conditioned media were normalized
relative to the concentration of the same biomarker in conditioned
media from control cultures (2D culture without biomaterial) and
expressed as a unitless ratio.
Proteomic profiling. Protein from three independent replicates was
extracted from cell/biomaterial composites and pooled for analysis
by 2D gel electrophoresis. All reagents were from Invitrogen.
Isoelectric focusing (IEF) was conducted by adding 30 .mu.g of
protein resuspended in 200 .mu.l of ZOOM 2D protein solubilizer #1
(Cat #ZS10001), ZOOM carrier ampholytes pH 4-7 (Cat #ZM0022), and
2M DTT (Cat #15508-013) to pH 4-7 ZOOM IEF Strips (Cat #ZM0012).
Following electrophoresis for 18 hours at 500V, IEF strips were
loaded onto NuPAGE Novex 4-12% Bis-Tris ZOOM IPG well gels (Cat
#NP0330BOX) for SDS-PAGE separation and electrophoresed for 45 min
at 200V in MES buffer (Cat #NP0002). Proteins were visualized using
SYPRO Ruby protein gel stain (Cat #S-12000) according to the
manufacturer's instructions.
Confocal microscopy. NKA Constructs prepared from human or rat UNFX
or B2 cells were matured for 3 days and then fixed in 2%
paraformaldehyde for 30 minutes. Fixed NKA Constructs were blocked
and permeabilized by incubation in 10% goat serum (Invitrogen) in
D-PBS (Invitrogen)+0.2% Triton X-100 (Sigma) for 1 hour at room
temperature (RT). For immunofluorescence. NKA Constructs were
labeled with primary antibodies (Table 15.2) at a final
concentration of 5 .mu.g/ml overnight at RT. Labeled NKA constructs
were washed twice with 2% goat serum/D-PBS+0/2% Triton X-100 and
incubated with goat or rabbit TRITC conjugated anti-mouse IgG2A
(Invitrogen) secondary antibody at 5 .mu.g/ml. For double labeling
with DBA (Dolichos biflorus agglutinin), NKA construct candidates
were further incubated with FITC conjugated DBA (Vector Labs)
diluted to 2 mg/ml in 2% goat serum/D-PBS+0.2% Triton X-100 for 2
hrs at RT.
TABLE-US-00014 TABLE 15.2 Antibody Source Manufacturer Catalog#
Target IgG1 ctrl Mouse BD 557273 Background control IgG ctrl goat
Invitrogen 026202 Background control IgG ctrl rabbit Invitrogen
026102 Background control N-Cadherin Mouse BD 610920 Proximal
tubules E-Cadherin Mouse BD 610182 Distal tubules Cubilin goat
Santa Cruz Sc-20609 Proximal tubules (A-20) GGT-1 Rabbit Santa Cruz
Sc-20638 Tubular epithelial Megalin Rabbit Santa Cruz Sc-25470
Proximal tubules
Samples were washed twice with D-PBS and optically sectioned using
a Zeiss LSM510 laser scanning confocal system (Cellular Imaging
Core, Wake Forest Baptist Medical Center) running LSM Image
software (Zeiss) or with a Pathway 855 confocal microscope (BD
Biosciences).
Analysis of TGF-.beta. mediated EMT in HK2 cells. HK2 cells (ATCC)
were cultured in 50:50 media in fibronectin or collagen (IV) coated
culture dishes (BD Biosciences). For EMT assays, HK2 cells were
seeded in 24-well collagen (IV) coated plates at 70-80% confluency
with 50:50 media or conditioned media collected from either two
dimensional (2D) human UNFX cultures or NKA Constructs made with
human UNFX that were matured for 3 days prior to media collection.
TGF-.beta. induction was initiated by adding 10 ng/ml to the
culture media 3 days prior to isolating RNA from the cells for the
EMT assay. EMT was monitored by qRT-PCR by analyzing the relative
expression of E-cadherin (an epithelial marker) and calponin
(mesenchymal marker) at the end of the three day incubation period.
RNA was prepared from harvested HK2 cells for TaqMan qRT-PCR
analysis as described above. Statistical analysis was done using
standard two tailed Student's t-test assuming equal variance for
each sample. Confidence intervals of 95% (p-value<0.05) and 99%
(p-value<0.01) were used to determine statistical
significance.
In vivo implantation of acellular biomaterials and NKA Constructs.
Lewis rats (6 to 8 weeks old) were purchased from Charles River
(Kalamazoo, Mich.). All experimental procedures were performed
under PHS and IACUC guidelines of the Carolinas Medical Center.
Under isoflurane anesthesia, female Lewis rats (approximately 2 to
3 months old) underwent a midline incision, and the left kidney was
exposed. 35 .mu.l of packed biomaterials (acellular biomaterial or
NKA Construct) were introduced by microinjection into the renal
parenchyma. Two injection trajectories were used: (i) from each
pole toward the cortex (referred to as cortical injection), or (ii)
from the renal midline toward the pelvis (referred to as medullary
injection). Rats were sacrificed at 1, 4, or 8 weeks
post-injection. No early deaths occurred. Study design for the
acellular implantation study is presented in Table 15.3 (ND=not
done).
Renal Histology. Representative kidney samples were collected and
placed in 10% buffer formalin for 24 hours. Sections were
dehydrated in ascending grades of ethanol and embedded in paraffin.
Sections (5 .mu.m) were cut, mounted on charged slides, and
processed for hematoxylin and eosin (H&E), Masson's trichrome
and Periodic Acid Schiff (PAS) staining in accordance with standard
staining protocols (Prophet et al., Armed Forces Institute of
Pathology: Laboratory methods in histotechnology. Washington, D.C.:
American Registry of Pathology; 1992). Digital microphotographs
were captured at total magnification of .times.40, .times.100 and
.times.400 using a Nikon Eclipse 50i microscope fitted with a
Digital Sight (DS-U1) camera. Renal morphology changes were
assessed by commonly used (Shackelford et al. Toxicol Pathol
30(1):93-96; 2002) severity grade schemes (grades 1, 2, 3, 4), to
which descriptive terms (minimal, mild, moderate, marked/severe)
were applied to describe the degree of glomerulosclerosis, tubular
atrophy and dilatation, tubular casts, and interstitial fibrosis,
and inflammation observed.
TABLE-US-00015 TABLE 15.3 Study design for evaluating acellular
biomaterials in healthy adult Lewis rat kidneys Time in vivo
Biomaterial: 1 week 4 weeks PCL Beads n = 1 n = 1 Gelatin Beads n =
1 ND Gelatin Particles n = 1 n = 1 HA/Gelatin Particles n = 2 ND HA
Particles n = 1 n = 1 PLGA Particles n = 1 ND PLGA Beads n = 1
ND
Results
Response of mammalian kidney tissue to injection of biomaterials
into the renal parenchyma. Biomaterials were analyzed for potential
use in renal cell/biomaterial composites by direct injection into
healthy rat kidneys (Table 15.3). Tissue responses were evaluated
by measuring the degree of histopathology parameters (inflammation,
fibrosis, necrosis, calcification/mineralization) and
biocompatibility parameters (biomaterial degradation,
neo-vascularization, and neo-tissue formation) at 1 and 4 weeks
post-injection.
FIGS. 19A-B show in vivo evaluation of biomaterials at 1 week
post-implantation, Trichrome X10 low power image of kidney cross
section showing biomaterial aggregate. Trichrome X40: Close-up of
biomaterial aggregate. H&E X400: High magnification image of
biomaterial aggregate to evaluate extent of cell/tissue
infiltration. Each kidney was injected at two locations as
described in Materials and Methods. At 1 week post-implantation,
the host tissue responses elicited by each biomaterial tested were
generally similar; however, gelatin hydrogels appeared to elicit
less intense histopathological and more biocompatible
responses.
FIG. 19C shows in vivo evaluation of biomaterials at 4 weeks
post-implantation. At 4 weeks post-implantation, the severity of
histopathology parameters in tissues injected with HA or gelatin
particles were qualitatively reduced compared to 1 week
post-implantation. Gelatin particles were nearly completely
resorbed and less giant cell reaction was observed than in tissues
that received HA particles. In most cases where biomaterials were
injected via the medullary injection trajectory (e.g., deeper into
the medulla/pelvis), undesirable outcomes including obstruction
leading to hydronephrosis, inflammatory reactions of greater
severity, and renal arteriolar and capillary micro-embolization
leading to infarction was observed (data not shown).
Assessing functional phenotype of therapeutically-relevant renal
cell populations with biomaterials. Therapeutically-relevant renal
cell populations (UNFX) that extended survival and increased renal
function in a rodent model of chronic kidney disease after direct
injection into renal parenchyma have been characterized (Presnell
et al, WO/2010/056328; Kelley et al, supra 2010) and methods for
their isolation, characterization, and expansion have been
developed and translated across multiple species (Presnell al. 2010
supra). To assess whether UNFX cells adhere to, remain viable, and
retain a predominantly tubular, epithelial phenotype when
incorporated into NKA Constructs, transcriptomic, secretomic,
proteomic, and confocal immunofluorescence microscopy analyses were
conducted on NKA Constructs produced from UNFX cells and various
biomaterials.
Adherence and viability. Canine-derived UNFX cells were seeded with
gelatin beads, PCL beads, PLGA beads, HA particles, and HA/gelatin
particles as described (3 NKA Constructs per biomaterial). Cell
distribution and viability were assessed one day after seeding by
live/dead staining
FIGS. 20A-D show live/dead staining of NKA constructs seeded with
canine UNFX cells (A=gelatin beads; B=PCL beads; C=HA/gelatin
particles; D=HA particles). Green indicates live cells; red
indicates dead cells. (A) Gelatin beads; (B) PCL beads; (C)
HA/gelatin particles; and (D) HA particles. Viable cells may be
observed on all hydrogel-based NKA Constructs.
UNFX cells adhered robustly to naturally-derived, hydrogel-based
biomaterials such as gelatin beads and HA/gelatin particles (black
arrows in A, D), but showed minimal adherence to synthetic. PCL (B)
or PLGA beads (not shown). Cells did not adhere to HA particles (C)
but showed evidence of bioresponse (i.e., spheroid formation).
Functional viability of the seeded UNFX cells on hydrogel-based NKA
Constructs was confirmed by assaying for leucine aminopeptidase, a
proximal tubule-associated hydrolase (data not shown).
Transcriptomic profiling. The gene expression profiles of human
UNFX cells in hydrogel-based NKA Constructs (3 NKA Constructs per
biomaterial) and parallel 2D cultures of UNFX cells were compared
by quantitative transcriptomic analysis.
FIGS. 20E-G show transcriptomic profiling of NKA constructs. TC:
primary human UNFX cells cultured in 2D. Gelatin: NKA Construct
composed of human UNFX cells and gelatin hydrogel. HA-Gel: NKA
Construct composed of human UNFX cells and HA/gelatin particles.
qRT-PCR data presented in graphical and tabular format. Transcripts
examined fell into four principal categories: (i) Tubular:
aquaporin 2(AQ2), E-cadherin (ECAD), erythropoietin (EPO),
N-cadherin (NCAD), Cytochrome P450, Family 24, Subfamily A,
Polypeptide 1--aka Vitamin D 24-Hydroxylase (CYP), cubilin,
nephrin; (ii) Mesenchymal: calponin (CNN1), smooth muscle myosin
heavy chain (SMNINC); (iii) Endothelial: vascular endothelial
growth factor (VEGF), platelet endothelial cell adhesion molecule
(PECAM); and (iv) Glomerular: podocin. Overall, tubular marker
expression was comparable between hydrogel-based NKA Constructs and
2D UNFX cultures. Similarly, endothelial markers (VEGF and PECAM)
were comparable. In contrast, the glomerular marker podocin
exhibited significant variation among NKA Constructs. Podocin
levels in HA/gelatin-based NKA Constructs were most comparable with
those observed in 2D UNFX cultures. Interestingly, mesenchymal
marker (CNN1 and SMMHC) expression was significantly down-regulated
(p<0.05) in hydrogel-based NKA Constructs relative to 2D UNFX
cultures, suggesting that fibroblastic sub-populations of UNFX may
not propagate as well in the hydrogel-based NKA Constructs in the
renal media formulation.
Secretomic profiling. NKA Constructs were produced with human UNFX
and B2 cells and gelatin or HA/gelatin hydrogel (one NKA Construct
per biomaterial per cell type=4 NKA Constructs total).
FIGS. 21A-B show the secretomic profiling of NKA Constructs. Data
is presented as a 3D:2D ratio. NKA Constructs were produced from
human UNFX or B2 cells and gelatin (Hydrogel 1) or HA/gelatin
(Hydrogel 2) hydrogels as described in Materials and Methods.
Secretomic profiling was performed on conditioned media from NKA
Constructs matured for 3 days and compared with parallel 2D
cultures of human UNFX or B2 cells by calculating the ratio of
analyte expression of NKA Constructs (three-dimensional, or 3D,
culture) to 2D culture (3D:2D ratio). For each of the three NKA
Constructs seeded with UNFX cells, the 3D:2D ratios were at or
close to 1, suggesting that the seeding process and 3 days of
maturation on these biomaterials had little impact on the
secretomic profile of UNFX cells. For NKA Constructs seeded with B2
cells, a similar result of a 3D:2D ratio at or near 1 was observed,
providing additional evidence that the seeding process and 3 days
of maturation on these biomaterials had little impact on the
secretomic profile of therapeutically-relevant renal cells.
Proteomic profiling. Proteomic profiles of a given cell or tissue
are produced by separating total cellular proteins using 2D gel
electrophoresis and have been used to identify specific biomarkers
associated with renal disease (Vidal et al. Clin Sci (Lond)
109(5):421-430; 2005).
FIGS. 22A-B show proteomic profiling of NKA Constructs. NKA
Constructs were produced with human UNFX cells and biomaterials as
indicated. Proteins in total protein extracts were separated by 2D
gel electrophoresis as described in Materials and Methods. In this
experiment, proteomic profiling was used to compare protein
expression in human UNFX cells in NKA Constructs (gelatin or
HA/gelatin hydrogel-based, 3 NKA Constructs per biomaterial) and in
2D tissue culture. The proteome profiles of total protein isolated
from NKA Constructs or 2D cultures of UNFX cells were essentially
identical, providing additional evidence that the seeding process
and 3 days maturation on these biomaterials had little impact on
the proteomes expressed by UNFX cells.
Confocal microscopy. Retention of the tubular epithelial phenotype
of rat and human B2 cells (Presnell et al. 2010 supra) NKA
Constructs was evaluated by confocal imaging of established
biomarkers: FIGS. 23A-C show confocal microscopy of NKA Constructs,
Confocal microscopy of NKA Constructs produced with human (A) or
rat (B, C) B2 cells and gelatin hydrogel. (A) E-cadherin
(red--solid white arrows), DBA (green--dashed green arrows) and
gelatin hydrogel bead is visible with DIC optics. (B) DNA
visualized with DAPI staining (blue--solid white arrows) and each
of the following markers in green (dashed white arrows): IgG
control, N-cadherin, E-cadherin, cytokeratin 8/18/18, DBA. (C)
double-labeling images of markers and colors as indicated.
E-cadherin and DBA in human NKA Constructs and E-cadherin, DBA,
N-cadherin, cytokeratin 8/18/19, gamma glutamyl transpeptidase
(GGT-1), and megalin in rat NKA Constructs. Optical sectioning of
confocal images also allowed evaluation of the extent of cell
infiltration into the biomaterial after seeding and 3 days of
maturation. B2 cells in human and rat NKA Constructs exhibited
expression of multiple tubular epithelial markers. Optical
sectioning revealed minimal cell infiltration of the hydrogel
construct, with cells generally confined to the surface of the
biomaterial.
In vivo responses to implantation of NKA construct prototypes.
Based on the in vivo responses to biomaterial injection into renal
parenchyma and the in vitro phenotype and functional
characterization of UNFX and B2 cells in NKA Constructs described
above, gelatin hydrogel was selected to evaluate the in vivo
response to NKA Construct injection into renal parenchyma in
healthy Lewis rats. NKA Constructs were produced from syngeneic B2
cells and implanted into two animals, which were sacrificed at 1,
4, and 8 weeks post-implantation. All animals survived to scheduled
necropsy when sections of renal tissues were harvested, sectioned,
and stained with Trichrome, hematoxylin and eosin (H&E), and
Periodic Acid Schiff (PAS).
FIGS. 24A-B show in vivo evaluation of NKA Constructs at 1 and 4
weeks post-implantation. Trichrome X10 low power image of kidney
cross section showing biomaterial aggregate. Trichrome X40:
Close-up of biomaterial aggregate. H&E/PAS X400: High
magnification image of biomaterial aggregate to evaluate extent of
cell/tissue infiltration. Each kidney was injected at two locations
as described in Materials and Methods.
FIG. 24A shows in vivo evaluation of NKA Constructs at 1 week
post-implantation. At 1 week post injection, gelatin beads were
present as focal aggregates (left panel, circled area) of spherical
and porous material staining basophilic and surrounded by marked
fibro-vascular tissue and phagocytic multi-nucleated macrophages
and giant cells. Fibrovascular tissue was integrated within the
beads and displayed tubular epithelial components indicative of
neo-kidney tissue formation. Additionally, tubular and
vasculoglomerular structures were identified by morphology (PAS
panels).
FIG. 24B shows in vivo evaluation of NKA Constructs at 4 weeks
post-implantation. By 4 weeks post-injection, the hydrogel was
completely resorbed and the space replaced by progressive renal
regeneration and repair with minimal fibrosis (note the numerous
functional tubules within circled area of 4-week Trichrome
panel).
FIGS. 25A-D show in vivo evaluation of NKA Construct at 8 weeks
post-implantation. Trichrome X10 low power image of kidney cross
section showing biomaterial aggregate. Trichrome X40: Close-up of
biomaterial aggregate. H&E/PAS X400: High magnification image
of biomaterial aggregate to evaluate extent of cell/tissue
infiltration. (A) Moderate chronic inflammation (macrophages,
plasma cells and lymphocytes), moderate numbers of
hemosiderin-laden macrophages (chronic hemorrhage due to injection)
with marked fibrovascular response (blue stained by Masson's
trichrome--black arrows); (B) Higher magnification (trichrome
stained, .times.400) of boxed area of (A) showing regenerative
response induction consistent with neo-kidney tissue formation (C)
Representative of adjacent (normal) kidney parenchyma showing
typical cortical glomeruli morphology HE, .times.400); (D) HE
stained section, .times.400 comparing new glomeruli morphology
observed in treatment area vs. FIG. 25C.
FIGS. 25A-D show in vivo evaluation of NKA Construct at 8 weeks
post-implantation. At 8 weeks post-implantation, evidence of
neo-kidney like tissue formation was observed, consistent with
induction of early events in nephrogenesis. Comparison of the area
of regenerative induction (B, D) with adjacent cortical parenchyma
(C) showed presence of multiple S-shaped bodies and newly formed
glomeruli.
Effect of conditioned media from NKA Constructs on TGF-.beta.
induced EMT in HK2 cells. The development of tubulo-interstitial
fibrosis during the progression of CKD is associated with
TGF-.beta. mediated EMT of tubular epithelial cells (Zeisberg et
al. Am J Pathol 160(6):2001-2008; 2002). Also, attenuation of
TGF-.beta. pathways was observed in vivo in a rodent model of
progressive CKD where survival was extended and renal function
improved by treatment with UNFX and B2 cells (Presnell et al.
WO/2010/056328). The human proximal tubular cell line HK2 has been
well established as an in vitro model system to test the
stimulatory or inhibitory effects of small molecules or proteins on
TGF-.beta. induced EMT (Dudas et al. Nephrol Dial Transplant
24(5):1406-1416; 2009; Hills et al. Am. J Physiol Renal Physiol
296(3):F614-621; 2009). To investigate a potential mechanism by
which NKA Constructs might affect renal tissue responses
post-implantation, conditioned medium collected from NKA Constructs
produced with UNFX cells and hydrogel was evaluated in the HK2 EMT
assay system.
FIG. 26 shows conditioned medium from NKA Constructs attenuates
TGF-.beta. induced EMT in HK2 cells in vitro. EMT is monitored by
quantitating the relative expression of ECAD (epithelial) and CNN1
(mesenchymal) markers. HK2 cells were cultured in 50:50 media
(Control and TGFB Control samples) or conditioned medium (CM) from
2D cultures of human UNFX cells (TC) or NKA Constructs produced
from human UNFX cells and either Gelatin or HA/Gelatin as
indicated. To induce EMT, 10 ng/ml TGF-.beta. was added to each
sample (except Control) for 3 days prior to assay. When HK2 cells
were cultured in 50:50 media (Control), ECAD (epithelial marker)
was expressed at higher levels than CNN1 (mesenchymal marker). When
TGF-.beta. is added to the media for 3 days (TGFB Control). ECAD
expression was significantly down-regulated with a concomitant
up-regulation of CNN1, consistent with induction of an EMT event.
Conditioned medium from 2D UNFX cell cultures significantly
(p<0.05 for both ECAD and CNN1) attenuated the EMT response of
HK2 cells to TGF-.beta. (TC CM). Conditioned medium from NKA
Constructs (Gelatin CM and HA/Gelatin CM) also attenuated the EMT
response to TGF-.beta.; however the overall effect was less than
that observed with conditioned medium from 2D UNFX cell cultures
(significant--p<0.05--for ECAD with both NKA Constructs and
trending toward control though not statistically significant for
CNN1). Additional mesenchymal markers were screened and yielded
similar results (data not shown). These data suggest that NKA
Constructs could potentially affect TGF-.beta. pathways associated
with tubulo-interstitial fibrosis in vivo in a manner similar to
that observed with cell-based treatment (Presnell et al.
WO/2010/056328). These data also suggest that the in vitro EMT
assay has potential application for screening/optimizing/monitoring
the biotherapeutic efficacy of NKA Constructs if in vivo responses
can be demonstrated to have a statistically significant association
with in vitro EMT responses, thereby potentially reducing the need
for time consuming and expensive in vivo assays.
This study investigated the responses of mammalian renal parenchyma
to implantation of synthetic and natural biomaterials, both
acellular and as bioactive renal cellibiomaterial composites (i.e.,
NKA Constructs). A combination of in vitro functional assays and in
vivo regenerative outcomes were analyzed to functionally screen
candidate biomaterials for potential incorporation into a NKA
construct prototype. Implantation of acellular hydrogel-based
biomaterials into renal parenchyma (FIG. 19) was typically
associated with minimal fibrosis or chronic inflammation and no
evidence of necrosis by 4 weeks post-implantation. Moderate
cellular/tissue in-growth and neo-vascularization was observed,
with minimal remnant biomaterial. Based on these in vivo data,
hydrogel-based biomaterials were selected to produce NKA Constructs
with which to evaluate in vitro biofunctionality and in vivo
regenerative potential. In vitro confirmation of material
biocompatibility was provided through live/dead analysis of NKA
Constructs (FIG. 20). Gelatin-containing hydrogels were associated
with robust adherence of primary renal cell populations. Phenotypic
and functional analysis of NKA Constructs produced from bioactive
primary renal cell populations (UNFX or B2) and hydrogel
biomaterials was consistent with continued maintenance of a tubular
epithelial cell phenotype. Transcriptomic, secretomic, proteomic,
and confocal microscopy analyses of NKA Construct confirmed no
significant differences relative to primary renal cells seeded in
2D culture. Finally, implantation of hydrogel-based NKA construct
into the renal parenchyma of healthy adult rodents was associated
with minimal inflammatory and fibrotic response and regeneration of
neo-kidney like tissue by 8 weeks post-implantation.
Taken together, these data provide evidence suggesting that a
regenerative response was induced in vivo by NKA Constructs. These
studies represent the first in vivo, intra-renal investigations of
the biological response of mammalian kidney to implantation of a
therapeutically-relevant primary renal cell/biomaterial composite.
Observed results are suggestive that NKA Constructs have the
potential to both facilitate regeneration of neo-kidney tissue and
attenuate non-regenerative (e.g., reparative healing)
responses.
Bioresponse of Mammalian Kidney to Implantation of Polymeric
Materials. In another study, host tissue responses to intra-renal
injection of natural and synthetic biomaterials in rodent kidney
were investigated to evaluate candidate biomaterials for forming
cell/biomaterial composites with bioactive renal cell populations
(Presnell et al. supra 2010). Methods: Natural biomaterials
included gelatin and hyaluronic acid (HA). Synthetic biomaterials
included polycaprolactone (PCL) and poly-lactic-co-glycolic acid
(PLGA). Candidate biomaterials were evaluated in two discrete
physical conformations: homogenous, spherical beads or heterogenous
and non-uniform particles, PCL and PLGA beads were prepared using a
modified double emulsion (water/oil/water) solvent extraction
method. Gelatin beads were purchased (Cultispher-S.RTM.,
Sigma-Aldrich, St. Louis, Mo.). PLGA particles were prepared using
a solvent casting porogen leaching technique; gelatin and HA
particles were prepared from cross-linked, lyophilized foam. Two
injections of 35 .mu.l of loosely packed biomaterials were
delivered to the left kidney parenchyma of 3 month old Lewis rats,
Histopathologic evaluation of formalin-fixed sections of kidney
tissue at 1 and 4 weeks post-injection was conducted using a
semi-quantitative grading severity scale from 0 (absent) to 4
(marked) of inflammation, tissue/cellular in-growth,
neo-vascularization, material degradation, and fibro-cellular
responses. Overall scores were calculated as the ratio of %
positive to % negative response (the higher the overall score the
superior outcome).
Results. Histopathologic evaluation performed on biomaterial
candidates--representative 40.times. images of kidneys harvested 1
week post-implantation, sections stained with Masson's Trichrome
(data not shown). Materials composed of polymers of natural origin,
such as gelatin and HA were associated with milder fibro-cellular
response and chronic inflammation, and greater cellular in-growth,
neo-vascularization, biomaterial degradation, and necessary
inflammation required for tissue healing and integration when
compared to the synthetic biomaterials, such as PLGA and PCL
(organized fibrous encapsulation). Summary of histopathologic
evaluation scoring. Scores were averaged by material composition
(mean.+-.SD). The synthetic materials (PLGA and PCL) scored the
lowest, and gelatin materials generally scored higher than HA
materials. This trend is most pronounced at the 4 week time point.
Due to factors unrelated to the material injections, not all the
samples tested at 1 week were available for analysis at 4 weeks.
The number of samples that are included in the gelatin, HA, and
synthetic groups are 3, 4, 3 at 1 week and 2, 3, 1 at 4 weeks,
respectively.
Biomaterials of natural origin (e.g., gelatin or HA) delivered by
injection to healthy renal parenchyma elicited tissue responses
were less pathologic at 4 weeks post-injection than those of
synthetic origin as measured by semi-quantitative histopathologic
evaluation.
Example 16--Hypoxic Exposure of Cultured Human Renal Cells Induces
Mediators of Cell Migration and Attachment and Facilitates the
Repair of Tubular Cell Monolayers In Vitro
The role of oxygen tension in the isolation and function of a
selected population of renal epithelial cells (B2) with
demonstrated therapeutic function in models of chronic kidney
disease (CKD) was investigated. This study examined whether low
oxygen exposure during processing alters composition and function
of selected human selected renal cells (SRCs) or bioactive renal
cells (BRCs). Upon exposure to 2% Oxygen, the following was
observed: an alteration of the distribution of cells across a
density gradient (see Presnell et al. WO 10/056328 incorporated
herein by reference in its entirety), improvement in overall
post-gradient yield, modulation of oxygen-regulated gene expression
(previously reported in Kelley et al. supra (2010)), increased
expression of erythropoietin, VEGF, HIF1-alpha, and KDR(VEGFR2).
In-process exposure to low oxygen enhances the ability of selected
bioactive renal cells to repair/regenerate damaged renal
tubules.
FIG. 27 depicts the procedure for exposing cells to low oxygen
during processing. FIG. 28 shows that upon exposure to 2% Oxygen,
the following was observed: alters distribution of cells across a
density gradient, improves overall post-gradient yield. Hypoxic
exposure (<3%) increased recovery of cultured human CKD-derived
renal cells from iodixanol-based density gradients relative to
atmospheric oxygen tension (21%) (96% vs. 74%) and increased the
relative distribution of selected cells (B2) into high-density
(>9% iodixanol) fractions (21.6% vs. 11.2%).
Competitive in vitro assays demonstrated that B2 cells pre-exposed
for 24 hours to hypoxic conditions were more proficient in
repairing damaged renal proximal tubular monolayer cultures than B2
cells cultured at 21% oxygen tension, with 58.6%.+-.3% of the
repair occurring within two hours of injury.
FIG. 29A depicts an assay developed to observe repair of tubular
monolayers in vitro. 1. Cells are labeled with fluorescent dyes (2%
oxygen, 21% oxygen, and HK2 tubular cells). 2. The tubular cell
monolayer was established and wounded. 3. Oxygen-exposed labeled
cells are added (2% and 21% exposed cells). They are seeded equally
at 20,000/cm2. Culturing is in serum-free media at 5% O2 for 24
hrs. 4. Cells that repair wounding are quantified. FIG.
29B--Quantitative Image Analysis (BD Pathway 855 BioImager)--red
circles=cells cultured 2% O2, blue circles=21% O2. FIG. 29C--it was
observed that 2% oxygen-induced cells attached more rapidly (2 hrs)
and sustained a mild advantage for 24 hrs. Cells induced with 2%
oxygen were more proficient at repair of tubular epithelial
monolayers.
FIG. 30A depicts an assay developed to observe repair of tubular
monolayers in vitro. 1. Cells were labeled with fluorescent dyes.
2. The tubular cell monolayer was established on the bottom of 8
.mu.m pore size transwell inserts and wounded. 3. The inserts are
flipped and oxygen-exposed labeled cells are added (2% and 21%
exposed cells). They are seeded equally at 50,000 cm2. Culturing is
in serum-free media at 5% O2 for 24 hrs. 4. Cells that repair
wounding are quantified.
FIG. 30B shows that the induction of cells with 2% Oxygen enhanced
the migration and wound repair compared to un-induced (21% oxygen).
FIG. 30C plots the % of migrated cells against the migration time.
The average number of cells and average percentage of cells are
provided in Table 16.1.
Hypoxia also induced mRNA expression of CXCR4, MMP9, ICAM1, and
dystroglycan; genes that mediate cell migration and attachment.
Focal accumulation of MMP9 and an increase in Connexin 43
aggregates on the cells' plasma membrane was confirmed by
immunocytochemistry.
FIG. 31A shows that osteopontin is secreted by tubular cells and is
upregulated in response to injury (Osteopontin Immunocytochemistry:
Hoechst nuclear stain (blue), Osteopontin (Red), 10.times.).
Osteopontin is a secreted phosphorylated glycoprotein (Kelly et al.
J Am Soc Nephrol, 1999). Osteopontin is expressed in kidney tubules
and is involved in adhesion and migration. Osteopontin is
upregulated by injury in established tubular cell monolayers as
shown by immunofluorescence (FIG. 31A) and ELISA (FIG. 31B).
TABLE-US-00016 TABLE 16.1 Quantitative image analysis using Simle
PCI 3 hr 24 hr N = 3 Average # cells Average % Average # cells
Average % 2% O.sub.2 26.33 61.51% 117.67 60.35% 21% O.sub.2 16.67
38.49% 76.33 39.65%
FIG. 32A shows that the migratory response of cells is mediated in
part by osteopontin (Green=migrated cells (5.times.)). FIG. 32B
shows that neutralizing antibodies (NAb) to osteopontin reduce
renal cell migration response by 50%.
FIG. 33 shows that low-oxygen induction of cells modulates
expression of tissue remodeling genes. Caveolin 1 is a scaffolding
protein involved in modulation of integrin signaling. MMP9 is a
metalloproteinase that facilitates migration through extracellular
matrix degradation. ICAM1 is an intercellular adhesion molecule
associated with epithelial cell motility. CXCR4 is a chemokine
surface receptor that mediates cell migration.
FIG. 34 depicts a putative mechanism for low oxygen augmentation of
bioactivity of cells leading to renal regeneration.
Taken together, these results suggest that hypoxic exposure
facilitates the isolation of a specific renal cell subpopulation
with demonstrated bioactivity for repair of tubular injury in
vitro, and thus may potentially enhance the ability of these cells
to migrate and engraft into diseased tissue after in vivo delivery.
The SRCs demonstrated the ability to stabilize renal function and
enhance survival in a rodent model of progressive CKD. The low
oxygen levels (2% O2) provided the following: enhanced post-culture
recovery of selected regenerative cells; enhanced cellular
attachment and monolayer repair in response to tubular injury; and
stimulated cellular migration in response to tubular injury. In
addition, cellular migration and attachment were mediated in part
by osteopontin in vitro, low-oxygen upregulated integrins, secreted
proteins, and cell adhesion molecules which mediate tissue
remodeling, migration, and cell-cell communication.
Example 17--Urine-Derived Microvesicles
An analysis of the miRNAs and proteins contained within the luminal
contents of kidney derived microvesicles shed into the urine was
performed to determine whether they might be used as biomarkers for
assessing regenerative outcome. As excess microvesicles are shed
into the extracellular space, some fuse with neighboring cells
while others are excreted into the urine (Zhou et al. 2008. Kidney
Int. 74(5):613-621). These urinary microvesicles now become
excellent biomarkers for assay development in order to better
understand treatment outcomes.
The ZSF1 rodent model of metabolic disease with chronic progressive
renal failure was used. B2+B4 cells were injected into the renal
parenchyma of ZSF1 animals. Healthy animals and PBS vehicle were
used as controls. Urine-derived vesicles were analyzed at different
time points as summarized below. 1: ZSF1 animal--PBS vehicle
injected; urine collected 197 days after injection 2: ZSF1
animal--PBS vehicle injection; urine collected 253 days after
injection 3: ZSF1 animal--B2+B4 fraction injected; urine collected
197 days after injection 4: ZSF1 animal--B2+B4 fraction injected;
urine collected 253 days after injection 5. ZSF1 animal--no
injection; urine collected on day 197 of the study 6. ZSF1
animal--no injection; urine collected on day 253 of the study 7.
Healthy animal--no injection; urine collected on day 197 of the
study 8. Healthy animal--no injection; urine collected on day 253
of the study Urine was collected from the test animals on day 197
and about 253 days after treatment. Microvesicles were recovered
from the urine by standard methods known in the art (for example,
see Zhou et al. Kidney Int. 2008 September; 74(5): 613-621). As
shown by standard Western blotting in FIG. 35, microvesicles
recovered from the urine of treated animals (lanes 3-4) showed an
increase in proteins associated with progenitor cells (CD133 &
WNT7A) when compared to either vehicle treated (lanes 1-2) or
untreated controls (lanes 5-8). In fact, microvesicles were only
recovered from the urine of diseased animals (lanes 1-6), not
healthy controls (lanes 7-8), as indicated by expression of the
microvesicle specific protein CD63 (FIG. 35). The CD133-containing
microvesicles appear to be prominosomes shed from kidney cells.
Both CD133 and WNT7A have been associated with regeneration and
stem cell division (Romagnani P and Kalluri R. 2009. Fibrogenesis
Tissue Repair. 2(1):3; Lie et al. 2005. Nature. 437(7063):1370-5;
Willert et al, 2003. Nature. 423(6938):448-52; Li et al. 2009. Am J
Physiol Renal Physiol. 297(6):F1526-33). Taken together, this
supports targeting proteins expressed in microvesicles as
biomarkers for assay development designed to monitor
regeneration.
miRNA microarrays and RT-PCR. Microarray and RT-PCR analysis of
miRNA from urine-derived vesicles was performed by standard methods
known in the art (for example, see Wang et al. supra 2010). In
addition to proteins, miRNAs were found within the contents of the
isolated microvesicles. Table 17.1 provides examples of miRNAs that
were found to be increased with treatment.
TABLE-US-00017 TABLE 17.1 miRNA RQ value miRNA RQ value miRNA RQ
value miR-15b 6.5206 miR-21 6.4755 miR-30a 6.0002 miR-30a* 2.4666
miR-30b-5p 9.8833 miR-30c 6.1688 miR-30d 5.9176 miR-30d* 4.1482
miR-30e 8.0836 miR-30e* 2.1622 miR-141 5.1515 miR-146a 2.3054
miR-151 3.4462 miR-200a 9.3340 miR-200c 8.0278 miR-429 9.7136
The change in was analyzed in ZSF1 animals treated with B2+B4 over
time (day 197 and day 253). A fold change was observed for the
following miRNAs:
TABLE-US-00018 miR-370 miR-1224 miR-598-5p miR-362 miR-22 miR-540
miR-300-3p miR-154 miR-345-5p miR-206 miR-375 miR-29c miR-222
miR-298 miR-215 miR-433 miR-671 miR-138* let-7b miR-21 miR-182
let-7c let-7e miR-147 miR-497 let-7f miR-664 let-7b* miR-125a-5p
miR-339-5p miR-24-2* miR-194 miR-743b miR-770 miR-152 miR-96
miR-124 miR-128 miR-203 miR-485 miR-423 miR-130a miR-760-5p miR-92b
miR-346 miR-296 miR-139-3p miR-674-3p miR-764 miR-667 miR-140*
miR-221 miR-98 miR-192 miR-883 miR-181d miR-409-3p miR-15b miR-500
miR-106b* miR-143 miR-103 let-7d let-7a miR-99b* miR-28* miR-339-3p
miR-935 miR-219-2-3p miR-484 miR-674-5p miR-148b-3p miR-742
miR-743a miR-23b miR-101b miR-352 miR-195 miR-425 miR-326 miR-30d
miR-31 miR-181c miR-301b miR-29b miR-504 miR-320 let-7e* miR-322*
miR-652 miR-345-3p miR-7a miR-322 miR-193 miR-532-3p miR-429
miR-207 miR-29a miR-291a-5p miR-501 miR-130b miR-26a miR-125b-3p
miR-328 miR-141 miR-185 miR-100 miR-543 miR-27b miR-874 miR-210
miR-200c miR-200a miR-30a* miR-431 miR-30c miR-365 miR-16 miR-99b
miR-378 miR-23a miR-212 miR-196b miR-330* let-7i miR-27a*
miR-344-5p miR-30a miR-361 miR-347 miR-199a-5p miR-10b miR-125b*
miR-25 miR-466c miR-30b-5p miR-99a* miR-342-3p miR-218 miR-28
miR-216a miR-99a miR-30d* miR-101a* miR-488 miR-151* miR-24 miR-493
miR-344-3p miR-151 miR-9* miR-126* miR-205 miR-92a miR-145
miR-17-5p miR-125b-5p miR-20b-5p miR-134 miR-183 miR-294 miR-296*
miR-708 miR-489 miR-26b* miR-191 miR-483 miR-30e* miR-181b miR-132
miR-382 miR-186 miR-505 miR-410 miR-872 miR-7a* miR-327 miR-188
let-7i* miR-133b miR-22* miR-21* miR-142-3p miR-27a miR-331
miR-324-5p miR-7b miR-434 miR-125a-3p miR-598-3p miR-106b let-7d*
miR-490 miR-30e miR-19b miR-675 miR-350 miR-223 miR-10a-5p miR-34a
miR-374 miR-184 miR-219-1-3p miR-127 miR-26b miR-871 miR-107
miR-25* miR-93 miR-34c* miR-330 miR-9 miR-672 miR-146a miR-532-5p
miR-873 miR-295 miR-181a miR-351 miR-196a miR-760-3p miR-292-5p
miR-34b miR-140 miR-343 miR-129 miR-29a* miR-449a miR-196c miR-138
miR-323 miR-17-3p miR-193* miR-29b-2* miR-133a miR-10a-3p miR-539
miR-19a miR-877 miR-465 miR-211 miR-29c* miR-214 miR-20a miR-503
miR-541 miR-34c miR-148b-5p miR-761
miRNA levels were analyzed in ZSF1 animals treated with B2+B4 (day
253) and compared to the miRNA levels in ZSF1 animals treated with
PBS vehicle (day 253). A fold change was observed for the following
miRNAs:
TABLE-US-00019 miR-24 miR-16 miR-10b miR-195 let-7d* miR-365
miR-871 miR-30e miR-431 miR-30b-5p miR-200c miR-29c miR-19b
miR-292-5p miR-15b miR-99a miR-152 miR-21 miR-429 let-7i
miR-125b-5p let-7f miR-351 miR-30c miR-200a miR-434 miR-30a
miR-324-5p miR-26b miR-503 miR-10a-5p miR-489 miR-26a miR-148b-3p
miR-186 miR-30d miR-100 miR-191 miR-743b miR-9 miR-30d* miR-184
let-7e miR-148b-5p miR-375 miR-23a miR-138* let-7b miR-322* miR-465
miR-133a miR-96 miR-129 miR-345-5p miR-106b miR-344-3p miR-342-3p
miR-23b miR-330 miR-34a miR-27b miR-374 miR-7a miR-29a miR-10a-3p
miR-291a-5p let-7a miR-352 miR-34c miR-425 miR-181a miR-300-3p
miR-20a let-7i* miR-219-1-3p miR-22* miR-134 miR-30a* miR-98
miR-196a miR-125a-5p miR-25 miR-203 miR-323 miR-194 let-7c miR-327
let-7d miR-151 miR-883 miR-141 miR-20b-5p miR-541 miR-7a* miR-26b*
miR-708 miR-107 miR-140 miR-196c miR-93 miR-128 miR-216a miR-742
miR-222 miR-146a miR-505 miR-29a* miR-27a* miR-19a miR-872 miR-223
miR-17-5p miR-328 miR-106b* miR-743a miR-760-3p miR-466c miR-182
miR-295 miR-490 miR-378 miR-151* miR-326 miR-761 miR-138 miR-664
miR-103 miR-211 miR-29b miR-221 miR-339-5p miR-30e* miR-212 miR-382
miR-532-3p miR-27a miR-497 miR-199a-5p miR-154 miR-320 miR-760-5p
miR-127 miR-675 miR-346 miR-667 miR-105 miR-301a miR-92a miR-874
miR-488 miR-140* miR-500 miR-539 miR-322 miR-501 miR-132 miR-181b
miR-347 miR-126* miR-296* miR-338 miR-99b miR-339-3p miR-671
miR-125a-3p miR-28* miR-34c* miR-181d miR-24-2* miR-92b miR-543
miR-345-3p miR-29b-2* let-7b* miR-330* miR-298 miR-99b* miR-504
miR-139-3p miR-463 miR-449a miR-342-5p miR-146b miR-210 miR-877
miR-343 miR-188 miR-99a* miR-205 miR-410 miR-129* miR-23a*
miR-101a* miR-181c miR-674-5p miR-147 miR-21* miR-214 miR-192
miR-25* miR-540 miR-296 miR-143 miR-133b miR-196b miR-770 miR-29c*
miR-433 miR-652 miR-758 miR-17-3p miR-183 miR-362 miR-350 miR-22
miR-34b miR-142-3p miR-485 miR-409-3p miR-423 miR-935 miR-185
miR-101b miR-532-5p miR-344-5p miR-219-2-3p miR-764 miR-294 let-7e*
miR-193 miR-31 miR-484 miR-672 miR-125b-3p miR-1224 miR-325-5p
miR-204* miR-361 miR-206 miR-873 miR-711 miR-370 miR-218 miR-215
miR-130a miR-124 miR-301b miR-193* miR-130b
miRNA levels were analyzed in ZSR1 animals treated with B2+B4 (day
197) and compared to the miRNA levels in ZSF1 animals treated with
PBS vehicle (day 197). A fold change was observed for the following
miRNAs:
TABLE-US-00020 miR-143 miR-148b-3p miR-497 miR-370 miR-26a miR-425
miR-351 miR-186 miR-347 let-7a miR-24-2* miR-19a miR-152 miR-26b
miR-431 miR-141 miR-375 miR-17-5p let-7c let-7f miR-98 miR-222
miR-206 miR-434 miR-362 miR-29a miR-339-5p miR-200a miR-100 miR-296
miR-188 miR-29c miR-667 miR-429 miR-16 miR-181b miR-505 miR-96
miR-324-5p miR-21 miR-151 miR-30e let-7e miR-125a-3p miR-10a-5p
miR-182 miR-195 miR-125a-5p let-7b miR-210 miR-29b let-7i miR-742
miR-28* miR-200c miR-30d miR-106b* miR-99a miR-194 miR-30a miR-221
miR-433 miR-423 miR-30b-5p miR-23b miR-19b let-7d miR-124 miR-500
miR-103 miR-101b miR-92a miR-291a-5p miR-328 miR-193 miR-181d
miR-185 miR-342-3p miR-320 miR-743b miR-215 miR-345-3p miR-127
miR-132 miR-764 miR-345-5p miR-532-5p miR-191 miR-140* miR-203
miR-10b miR-20a miR-130b miR-298 miR-374 miR-449a miR-92b miR-664
miR-7a* miR-22 miR-877 miR-219-2-3p miR-330* miR-24 miR-23a miR-484
miR-205 miR-322 miR-339-3p miR-196b miR-181a miR-106b miR-126*
miR-219-1-3p miR-25 miR-20b-5p miR-30d* miR-326 miR-128 miR-301b
miR-129 miR-22* let-7e* miR-31 miR-196c miR-196a miR-34a miR-192
miR-9 miR-652 miR-151* miR-27a* miR-15b miR-134 miR-488 miR-130a
miR-214 miR-183 miR-378 miR-674-5p miR-26b* miR-30c miR-125b-5p
miR-138 let-7d* miR-365 miR-382 miR-874 miR-532-3p miR-760-3p
miR-485 miR-29c* let-7i* miR-93 miR-7a miR-184 miR-671 miR-147
miR-25* miR-99b* miR-27a miR-34c miR-139-3p miR-181c miR-30a*
miR-27b miR-99b miR-466c miR-21* miR-125b-3p miR-28 miR-142-3p
miR-344-5p miR-99a* miR-107 miR-493 miR-483 miR-148b-5p miR-17-3p
miR-223 miR-331 miR-7b miR-361 miR-218 miR-34b miR-503 miR-30e*
miR-193* miR-873 miR-330 miR-216a
The miRNAs listed in Table 17.1 provide examples of miRNAs that
have been implicated in processes relative to tissue regeneration.
miR-15b has been implicated in regulating apoptosis through BCL-2
and caspase regulation (Guo et al. 2009. J Hepatol. 50(4):766-78)
as well as cell cycle progression through the regulation of cyclins
(Xia et al. 2009. Biochem Biophys Res Commun. 380(2):205-10).
miR-21 was shown to inhibit apoptosis by modulating survival
pathways MAPK/ERK. The miR-30 family of miRNAs is critical for
podocyte structure and function suggesting that an increase maybe
necessary for glomerulargenisis. miR-141, 200a, 200c and 429 are
all involved in modulating epithelial to mesenchymal transition
(EMT) in response to TGF-.beta. signaling possibly reducing
fibrosis (Saal et al. 2009. Curr. Opin. Nephrol. Hypertens.
18:317-323). miR-146a and 151 have been implicated in NF.kappa.B
modulation thus potentially reducing the inflammatory response in
vivo (Taganov et al. 2006. Proc Natl Acad Sci USA. 103(33):12481-6;
Griffiths-Jones et al. 2006. NAR. 34 Database Issue: D140-D144).
Collectively, these miRNAs regulate processes related to a
successful regenerative outcome; thus making them candidate
biomarkers for assay development. Overall, this data supports the
concept that urinary microvesicles and/or their luminal contents
are viable targets for regenerative assays as they contain proteins
and miRNAs capable of modulating multiple pathways including: TGF
.beta.-1, NF.kappa.B, apoptosis, cell division and pluripotency in
addition to providing practitioners with a non-invasive means of
monitoring treatment.
SEQUENCE LISTINGS
1
3121RNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1cgacucacau ccuacaaaug u 21226DNARattus
sp. 2ttcagagtgt agatgacttg tttaca 26327DNAHomo sapiens 3ttttggagtg
taggtgactt gtttact 27
* * * * *
References